Arabidopsis derived promoters for regulation of plant expression

ABSTRACT

The invention provides a method to identify a plurality of plant promoters having specified characteristics and promoters identified by the method. Also provided are transgenic plants comprising the genes identified by the methods of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/334,085 filed Jan. 18, 2006, which is a continuation-in-partof Ser. No. 09/887,567 filed Jun. 22, 2001, which claims the benefit ofthe filing date of U.S. Provisional Application Ser. No. 60/214,087filed Jun. 23, 2000, U.S. Provisional Application Ser. No. 60/213,848filed Jun. 23, 2000, and U.S. Provisional Application Ser. No.60/258,692, filed Dec. 29, 2000 under 35 U.S.C. §119(e), the entiredisclosures of which are incorporated by reference herein.

FIELD OF INVENTION

The present invention relates generally to the field of plant molecularbiology. More specifically, it relates to the regulation of geneexpression in plants.

BACKGROUND OF INVENTION

Manipulation of crop plants to alter and/or improve phenotypiccharacteristics (such as productivity or quality) requires theexpression of heterologous genes in plant tissues. Such geneticmanipulation relies on the availability of a means to drive and tocontrol gene expression as required. For example, genetic manipulationrelies on the availability and use of suitable promoters which areeffective in plants and which regulate gene expression so as to give thedesired effect(s) in the transgenic plant. It is advantageous to havethe choice of a variety of different promoters so that the most suitablepromoter may be selected for a particular gene, construct, cell, tissue,plant or environment. Moreover, the increasing interest incotransforming plants with multiple plant transcription units (PTU) andthe potential problems associated with using common regulatory sequencesfor these purposes merit having a variety of promoter sequencesavailable.

Promoters (and other regulatory components) from bacteria, viruses,fungi and plants have been used to control gene expression in plantcells. Numerous plant transformation experiments using DNA constructscomprising various promoter sequences fused to various foreign genes(for example, bacterial marker genes) have led to the identification ofuseful promoter sequences. It has been demonstrated that sequences up to500-1000 bases in most instances are sufficient to allow for theregulated expression of foreign genes. However, it has also been shownthat sequences much longer than 1000 bases may have useful featureswhich permit desirable, e.g., high, levels of gene expression intransgenic plants.

One desirable source for promoters which have different expressionprofiles is plant genomic DNA. Plant development is preciselycoordinated and regulated through transcription and translation ofdifferent gene products in each cell. The expression level for each genepresent in a cell not only reflects the physiological status of thecell, but also determines the range of different functions the cell canperform. Identification of genes expressed constitutively, in a specificcell type or tissue, or at a specific developmental stage, and theanalysis of the abundance of the corresponding gene product can providevaluable insights into basic molecular processes and identity promoterswith desirable properties.

cDNA and high density oligonucleotide array technology allows analysisof mRNA transcripts of hundreds to thousands of genes in parallel(Schena et al., 1995; Chee et al., 1996; Lockhart et al., 1996; DeRisiet al., 1997; Lashkari et al., 1997). In some organisms with completedgenome sequences, such as yeast, global gene expression profiling at themRNA level becomes possible (DeRisi et al., 1997). Genome scaletranscription profiling enables not only parallel monitoring of geneexpression, but also a more subjective approach for gene discoverybecause objective selection of gene probes to be put on microarrays isnot required (Lockhart and Winzeler, 2000).

Microarray technology has been successfully developed for studying geneexpression in plants (Schena et al., 1995; Desprez et al., 1998; Yuan etal., 1998; Giege et al., 1998; Kehoe et al., 1999). The microarrays usedin those studies were cDNA microarrays on glass slides or filtermembranes (Duggan et al. 1999; Southern et al. 1999). The DNA probesoften consist of DNA fragments of expression sequence tags (ESTs) fromvarious Arabidopsis EST projects (i.e., Newman et al., 1994, Richmond etal., 2000, Schaffer et al., 2000). Microarrays with selected subsets ofgene probes (usually in the hundreds) has been used to examinedifferences in gene expression during organ development (Yuan et al.,1998; Aharoni et al., 2000), and has revealed genes that are correlatedor responsible for the defense response (Reymond et al., 2000).

There is, therefore, a great need in the art for the identification ofnovel sequences that can be used for expression of selected transgenesin economically important plants. More specifically, there is a need forthe systematic identification of genes that are expressed in aparticular manner, e.g., using microarray technology.

SUMMARY OF INVENTION

The present invention provides an isolated nucleic acid molecule(polynucleotide) having a plant nucleotide sequence that directstranscription of a linked nucleic acid segment in a plant or plant cell,e.g., a linked plant DNA comprising an open reading frame for astructural or regulatory gene. The nucleotide sequence preferably isobtained or isolatable from plant genomic DNA.

The present invention also provides an isolated nucleic acid moleculehaving a plant nucleotide sequence that directs constitutivetranscription of a linked nucleic acid segment in a host cell, e.g., aplant cell. The nucleotide sequence preferably is obtained or isolatablefrom plant genomic DNA. In particular, the nucleotide sequence isobtained or isolatable from an Arabidopsis gene which directsconstitutive transcription of a linked nucleic acid segment.

The present invention further provides an isolated nucleic acid moleculewhich comprises a plant nucleotide sequence that directs leaf-specific(i.e., preferential) transcription of a linked nucleic acid segment in aplant.

Thus, the presently disclosed subject matter provides in someembodiments isolated polynucleotides comprising a plant nucleotidesequence that directs transcription of an operatively linked nucleicacid segment in a plant cell. In some embodiments, the plant nucleotidesequences hybridize under high stringency conditions to a complement ofa sequence selected from the group consisting of SEQ ID NO:1-26. In someembodiments, the plant nucleotide sequence hybridizes under very highstringency conditions to the complement of SEQ ID NO: 1-26. In someembodiments, the plant nucleotide sequence is a functional fragment from25 to 2000 nucleotides in length.

The presently disclosed subject matter also provides expressioncassettes comprising the disclosed polynucleotides operatively linked toan open reading frame. In some embodiments, the open reading framecomprises a gene which, when transcribed at the direction of thepolynucleotide, imparts a phenotype selected from the group consistingof insect resistance, disease resistance, herbicide resistance, abioticstress resistance, a modified enzyme expression profile, a modified oilcontent, and a modified nutrient content.

The presently disclosed subject matter further provides transformedplants, the genome of which is augmented with one or more of thedisclosed expression cassettes. In some embodiments, the transformedplant is a monocot or a dicot plant. In some embodiments, thetransformed plant is a cereal plant. In some embodiments, the cerealplant is selected from the group consisting of maize, wheat, rice,sorghum, and barley. In some embodiments, the transformed dicot plant ofclaim 8 wherein the dicot is selected from the group consisting ofsoybean, cotton, canola, and sugarbeet

The presently disclosed subject matter also provides cells of thedisclosed transformed plants, which are characterized as having in theirgenomic DNAs the disclosed expression cassettes.

As described herein, GENECHIP® technology was utilized to discover genesthat are preferentially (or exclusively) expressed in various tissuesincluding root and leaf, as well as those that are constitutivelyexpressed, using labeled cRNA probes, determining expression levels bylaser scanning and generally selecting for expression levels thatwere >2 fold over the control. The Arabidopsis oligonucleotide probearray consists of probes from about 8,100 unique Arabidopsis genes,which covers approximately one third of the genome. This genome arraypermits a broader, more complete and less biased analysis of geneexpression. Using this approach, 51 genes were identified, theexpression of which was altered, e.g., elevated, in root tissues, and 92genes were identified, the expression of which was altered at least4-fold in leaf tissue. Similarly, 288 genes were identified that wereconstitutively expressed.

Generally, the promoters of the invention may be employed to express anopen reading frame from an insect resistance gene, a bacterial diseaseresistance gene, a fungal disease resistance gene, a viral diseaseresistance gene, a nematode disease resistance gene, a herbicideresistance gene, a gene affecting grain composition or quality, anutrient utilization gene, a mycotoxin reduction gene, a male sterilitygene, a selectable marker gene, a screenable marker gene, a negativeselectable marker, a gene affecting plant agronomic characteristics, oran environment or stress resistance gene, i.e., one or more genes thatconfer herbicide resistance or tolerance, insect resistance ortolerance, disease resistance or tolerance (viral, bacterial, fungal,oomycete, or nematode), stress tolerance or resistance (as exemplifiedby resistance or tolerance to drought, heat, chilling, freezing,excessive moisture, salt stress, or oxidative stress), increased yields,food content and makeup, physical appearance, male sterility, drydown,standability, prolificacy, starch properties or quantity, oil quantityand quality, amino acid or protein composition, and the like. By“resistant” is meant a plant which exhibits substantially no phenotypicchanges as a consequence of agent administration, infection with apathogen, or exposure to stress. By “tolerant” is meant a plant which,although it may exhibit some phenotypic changes as a consequence ofinfection, does not have a substantially decreased reproductive capacityor substantially altered metabolism.

In particular, root-specific promoters may be useful for expressingdefense-related genes, including those conferring insecticidalresistance and stress tolerance genes, e.g., salt, cold or droughttolerance, and genes for altering nutrient uptake, and leaf-specificpromoters may be useful for producing large quantities of protein, forexpressing oils or proteins of interest, genes for increasing thenutritional value of a plant, and for expressing defense-related genes(e.g., against pathogens such as a virus or fungus), including genesencoding insecticidal polypeptides. Constitutive promoters are usefulfor expressing a wide variety of genes including those which altermetabolic pathways, confer disease resistance, for protein production,e.g., antibody production, or to improve nutrient uptake. Constitutivepromoters may be modified so as to be regulatable, e.g., inducible.

The genes and promoters described hereinabove can be used to identifyorthologous genes and their promoters which are also likely expressed ina particular tissue and/or development manner. Moreover, the orthologouspromoters are useful to express linked open reading frames. In addition,by aligning the promoters of these orthologs, novel cis elements can beidentified that are useful to generate synthetic promoters. Hence, theisolated nucleic acid molecules of the invention include the orthologsof the Arabidopsis sequences disclosed herein, i.e., the correspondingnucleotide sequences in organisms other than Arabidopsis, including, butnot limited to, plants other than Arabidopsis, preferably cereal plants,e.g., corn, wheat, rye, turfgrass, sorghum, millet, sugarcane, soybean,barley, alfalfa, sunflower, canola, soybean, cotton, peanut, tobacco,sugarbeet, or rice. An orthologous gene is a gene from a differentspecies that encodes a product having the same or similar function,e.g., catalyzing the same reaction as a product encoded by a gene from areference organism. Thus, an ortholog includes polypeptides having lessthan, e.g., 65% amino acid sequence identity, but which ortholog encodesa polypeptide having the same or similar function. Databases suchGENBANK® may be employed to identify sequences related to theArabidopsis sequences, e.g., orthologs in cereal crops such as rice,wheat, sunflower or alfalfa.

Preferably, the promoters of the invention include a consecutive stretchof about 25 to 2000, including 50 to 500 or 100 to 250, and up to 1000or 1500, contiguous nucleotides, e.g., 40 to about 743, 60 to about 743,125 to about 743, 250 to about 743, 400 to about 743, 600 to about 743,of any one of SEQ ID NOs:1-26, or the promoter orthologs thereof, whichinclude the minimal promoter region.

In a particular embodiment of the invention said consecutive stretch ofabout 25 to 2000, including 50 to 500 or 100 to 250, and up to 1000 or1500, contiguous nucleotides, e.g., 40 to about 743, 60 to about 743,125 to about 743, 250 to about 743, 400 to about 743, 600 to about 743,has at least 75%, preferably 80%, more preferably 90% and mostpreferably 95% sequence identity with a corresponding consecutivestretch of about 25 to 2000, including 50 to 500 or 100 to 250, and upto 1000 or 1500, contiguous nucleotides, e.g., 40 to about 743, 60 toabout 743, 125 to about 743, 250 to about 743, 400 to about 743, 600 toabout 743, of any one of SEQ ID NOs:1-26, or the promoter orthologsthereof, which include the minimal promoter region.

In a preferred embodiment of the invention said consecutive stretch ofabout 25 to 2000, including 50 to 500 or 100 to 250, and up to 1000 or1500, contiguous nucleotides, e.g., 40 to about 743, 60 to about 743,125 to about 743, 250 to about 743, 400 to about 743, 600 to about 743,has at least 75%, preferably 80%, more preferably 90% and mostpreferably 95% sequence identity with a corresponding consecutivestretch of about 25 to 2000, including 50 to 500 or 100 to 250, and upto 1000 or 1500, contiguous nucleotides, e.g., 40 to about 743, 60 toabout 743, 125 to about 743, 250 to about 743, 400 to about 743, 600 toabout 743, of any one of SEQ ID NOs: 1-26, or the promoter orthologsthereof, which include the minimal promoter region.

Preferably, the nucleotide sequence that includes the promoter regionincludes at least one copy of a TATA box and, for leaf-specificexpression, preferably a light responsive element. Thus, the inventionprovides plant promoters, including orthologs of Arabidopsis promoterscorresponding to any one of SEQ ID NOs: 1-26 and orthologs thereof. Thepresent invention further provides a composition, an expression cassetteor a recombinant vector (e.g., a plasmid, phagemid, cosmid, virus,F-factor or phage) containing the nucleic acid molecule of theinvention, and host cells, e.g., a plant cell, comprising the expressioncassette or vector, e.g., comprising a plasmid. In particular, thepresent invention provides an expression cassette or a recombinantvector comprising a promoter of the invention linked to a nucleic acidsegment which, when present in a plant, plant cell or plant tissue,results in transcription of the linked nucleic acid segment.

In its broadest sense, the term “substantially similar” when used hereinwith respect to a nucleotide sequence means that the nucleotide sequenceis part of a gene which encodes a polypeptide having substantially thesame structure and function as a polypeptide encoded by a gene for thereference nucleotide sequence, e.g., the nucleotide sequence comprises apromoter from a gene that is the ortholog of the gene corresponding tothe reference nucleotide sequence, as well as promoter sequences thatare structurally related the promoter sequences particularly exemplifiedherein, i.e., the substantially similar promoter sequences hybridize tothe complement of the promoter sequences exemplified herein under highor very high stringency conditions. The term “substantially similar”thus includes nucleotide sequences wherein the sequence has beenmodified, for example, to optimize expression in particular cells, aswell as nucleotide sequences encoding a variant polypeptide having oneor more amino acid substitutions relative to the (unmodified)polypeptide encoded by the reference sequence, which substitution(s)does not alter the activity of the variant polypeptide relative to theunmodified polypeptide. In its broadest sense, the term “substantiallysimilar” when used herein with respect to polypeptide means that thepolypeptide has substantially the same structure and function as thereference polypeptide. The percentage of amino acid sequence identitybetween the substantially similar and the reference polypeptide is atleast 65%, 66%, 67%, 68%, 69%, 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, andeven 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, up to atleast 99%, wherein the reference polypeptide is an Arabidopsispolypeptide encoded by a gene with a promoter having any one of SEQ IDNOs:1-26. One indication that two polypeptides are substantially similarto each other, besides having substantially the same function, is thatan agent, e.g., an antibody, which specifically binds to one of thepolypeptides, specifically binds to the other.

Sequence comparisons may be carried out using a Smith-Waterman sequencealignment algorithm (see e.g., Waterman (1995)). The localS program,version 1.16, is preferably used with following parameters: match: 1,mismatch penalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2.Further, a nucleotide sequence that is “substantially similar” to areference nucleotide sequence hybridizes to the reference nucleotidesequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. with washing in 2×SSC, 0.1% SDS at 50° C., more desirably in 7%sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. withwashing in 1×SSC, 0.1% SDS at 50° C., more desirably still in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodium dodecyl sulfate(SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1%SDS at 50° C., more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C.

In one embodiment, the invention provides an expression cassette orvector containing an isolated nucleic acid molecule having a nucleotidesequence that directs root-specific, constitutive, or leaf-specifictranscription of a linked nucleic acid segment in a cell, whichnucleotide sequence comprises one or more of SEQ ID NOs: 1-26. Thisexpression cassette or vector may be contained in a host cell. Theexpression cassette or vector may augment the genome of a transformedplant or may be maintained extrachromosomally. The expression cassettemay be operatively linked to a structural gene, the open reading framethereof, or a portion thereof. The expression cassette may furthercomprise a Ti plasmid and be contained in an Agrobacterium tumefacienscell; it may be carried on a microparticle, wherein the microparticle issuitable for ballistic transformation of a plant cell; or it may becontained in a plant cell or protoplast. Further, the expressioncassette or vector can be contained in a transformed plant or cellsthereof, and the plant may be a dicot or a monocot. In particular, theplant may be a cereal plant.

The present invention further provides a method of augmenting a plantgenome by contacting plant cells with a nucleic acid molecule of theinvention, e.g., one having a nucleotide sequence that directsroot-specific, constitutive or leaf-specific transcription of a linkednucleic acid segment, so as to yield transformed plant cells; andregenerating the transformed plant cells to provide a differentiatedtransformed plant, wherein the differentiated transformed plantexpresses the nucleic acid molecule in the cells of the plant. Thenucleic acid molecule may be present in the nucleus, chloroplast,mitochondria and/or plastid of the cells of the plant. The presentinvention also provides a transgenic plant prepared by this method, aseed from such a plant and progeny plants from such a plant includinghybrids and inbreds. Preferred transgenic plants are transgenic maize,soybean, barley, alfalfa, sunflower, canola, soybean, cotton, peanut,sorghum, tobacco, sugarbeet, rice, wheat, rye, turfgrass, millet,sugarcane, tomato, or potato.

A transformed (transgenic) plant of the invention includes plants, forexample, a plant having transformed plant cells which cells contain anexpression cassette having a polynucleotide of the invention, or thegenome of which is augmented by a nucleic acid molecule of theinvention, or in which the corresponding gene has been disrupted, e.g.,to result in a loss, a decrease or an alteration, in the function of theproduct encoded by the gene, which plant may also have increased yieldsand/or produce a better-quality product than the corresponding wild-typeplant. The nucleic acid molecules of the invention are thus useful fortargeted gene disruption, as well as markers and probes.

The invention also provides a method of plant breeding, e.g., to preparea crossed fertile transgenic plant. The method comprises crossing afertile transgenic plant comprising a particular nucleic acid moleculeof the invention with itself or with a second plant, e.g., one lackingthe particular nucleic acid molecule, to prepare the seed of a crossedfertile transgenic plant comprising the particular nucleic acidmolecule. The seed is then planted to obtain a crossed fertiletransgenic plant. The plant may be a monocot or a dicot. In a particularembodiment, the plant is a cereal plant.

The crossed fertile transgenic plant may have the particular nucleicacid molecule inherited through a female parent or through a maleparent. The second plant may be an inbred plant. The crossed fertiletransgenic may be a hybrid. Also included within the present inventionare seeds of any of these crossed fertile transgenic plants.

The various breeding steps are characterized by well-defined humanintervention such as selecting the lines to be crossed, directingpollination of the parental lines, or selecting appropriate progenyplants. Depending on the desired properties different breeding measuresare taken. The relevant techniques are well known in the art and includebut are not limited to hybridization, inbreeding, backcross breeding,multiline breeding, variety blend, interspecific hybridization,aneuploid techniques, etc. Hybridization techniques also include thesterilization of plants to yield male or female sterile plants bymechanical, chemical or biochemical means. Cross pollination of a malesterile plant with pollen of a different line assures that the genome ofthe male sterile but female fertile plant will uniformly obtainproperties of both parental lines. Thus, the transgenic plants accordingto the invention can be used for the breeding of improved plant linesthat for example increase the effectiveness of conventional methods suchas herbicide or pesticide treatment or allow to dispense with saidmethods due to their modified genetic properties. Alternatively newcrops with improved stress tolerance can be obtained that, due to theiroptimized genetic “equipment”, yield harvested product of better qualitythan products that were not able to tolerate comparable adversedevelopmental conditions.

The present invention also provides a method to identify a nucleotidesequence that directs root-specific transcription of linked nucleic acidin the genome of a plant cell by contacting a probe of plant nucleicacid, e.g., cRNA, isolated from root as well as other tissues of aplant, with a plurality of isolated nucleic acid samples on one or more,i.e., a plurality of, solid substrates so as to form a complex betweenat least a portion of the probe and a nucleic acid sample(s) havingsequences that are structurally related to the sequences in the probe.Each sample comprises one or a plurality of oligonucleotidescorresponding to at least a portion of a plant gene. Then complexformation is compared between samples contacted with the root-specificprobe and samples contacted with a non-root specific probe so as todetermine which RNAs are expressed in root tissues of the plant. Theprobe and/or samples may be nucleic acid from a dicot or from a monocot.

The present invention also provides a method to identify a nucleotidesequence that directs constitutive transcription of nucleic acid in thegenome of a plant cell by contacting a probe of plant nucleic acid,e.g., cRNA, isolated from various tissues of a plant and at variousdevelopmental stages with a plurality of isolated nucleic acid sampleson one or more, i.e., a plurality of, solid substrates so as to form acomplex between at least a portion of the probe and a nucleic acidsample(s) having sequences that are structurally related to thesequences in the probe. Each sample comprises one or a plurality ofoligonucleotides corresponding to at least a portion of a plant gene.Complex formation is then compared to determine which RNAs are presentin a majority of, preferably in substantially all, tissues, in amajority of, preferably at substantially all, developmental stages ofthe plant. The probe and/or samples may be nucleic acid from a dicot orfrom a monocot.

The present invention also provides a method to identify a nucleotidesequence that directs transcription of nucleic acid in the genome of aplant cell in leaf tissue, by contacting a probe of plant nucleic acid,e.g., cRNA, isolated from leaf as well as other tissues of a plant witha plurality of isolated nucleic acid samples on one or more, i.e., aplurality of, solid substrates, so as to form a complex between at leasta portion of the probe and a nucleic acid sample(s) having sequencesthat are structurally related to the sequences in the probe. Each samplecomprises one or a plurality of, oligonucleotides corresponding to atleast a portion of a plant gene. Then complex formation is determined ordetected to identify which samples represent genes that are expressed inleaf. The probe and/or samples may be nucleic acid from a dicot or froma monocot.

The invention further includes a nucleotide sequence which iscomplementary to one (hereinafter “test” sequence) which hybridizesunder stringent conditions with a nucleic acid molecule of the inventionas well as RNA which is transcribed from the nucleic acid molecule. Whenthe hybridization is performed under stringent conditions, either thetest or nucleic acid molecule of invention is preferably supported,e.g., on a membrane or DNA chip. Thus, either a denatured test ornucleic acid molecule of the invention is preferably first bound to asupport and hybridization is effected for a specified period of time ata temperature of, e.g., between 55 and 70° C., in double strengthcitrate buffered saline (SC) containing 0.1% SDS followed by rinsing ofthe support at the same temperature but with a buffer having a reducedSC concentration. Depending upon the degree of stringency required suchreduced concentration buffers are typically single strength SCcontaining 0.1% SDS, half strength SC containing 0.1% SDS and one-tenthstrength SC containing 0.1% SDS.

A computer readable medium, e.g., a magnetic tape, optical disk, CD-ROM,random access memory, volatile memory, non-volatile memory, or bubblememory, containing one or more of the nucleotide sequences of theinvention as well as methods of use for the computer readable medium areprovided. This medium allows a nucleotide sequence corresponding to atleast one of SEQ ID NOs:1-26, or, e.g., a nucleic acid molecule that hasat least 70% nucleic acid sequence identity to at least one of SEQ IDNOs:1-26 or the complement thereof, to be used as a reference sequenceto search against a database. This medium also allows for computer-basedmanipulation of a nucleotide sequence corresponding to at least one ofSEQ ID NOs: 1-26.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, nucleic acid constructs areprovided that allow initiation of transcription in a “root-specific” or“leaf-specific” manner. Constructs of the invention comprise regulatedtranscription initiation regions associated with protein translationelongation, and the compositions of the present invention are drawn tonovel nucleotide sequences for root-specific as well as leaf-specificexpression. The present invention thus provides for isolated nucleicacid molecules comprising a plant nucleotide sequence that directstranscription of a linked nucleic acid fragment in a plant cell. Thus,these nucleotide sequences exhibit promoter activity in, for example,root or leaf tissues. Promoters may be obtained from other plant speciesby using the Arabidopsis promoter sequences described herein as probesto screen for homologous promoters in other plants by hybridizationunder low, moderate or stringent hybridization conditions. Regions ofthe promoter sequences of the present invention which are conservedamong species could also be used as PCR primers to amplify a segmentfrom a species other than Arabidopsis, and that segment used as ahybridization probe (the latter approach permitting higher stringencyscreening) or in a transcriptional assay to determine promoter activity.Moreover, the promoter sequences could be employed to identifystructurally related sequences in a database using computer algorithms.

These promoters are capable of driving the expression of a codingsequence in a target cell, particularly in a plant cell. The promotersequences and methods disclosed herein are useful in regulating in someembodiments tissue-specific expression of any heterologous nucleotidesequence in a host plant in order to vary the phenotype of that plant.These promoters can be used with combinations of enhancer, upstreamelements, and/or activating sequences from the 5′ flanking regions ofplant expressible structural genes. Similarly the upstream element canbe used in combination with various plant promoter sequences.

Also in accordance with the present invention, nucleic acid constructsare provided that allow initiation of transcription in a“tissue-independent,” “tissue general,” or “constitutive” manner.Constructs of this embodiment invention comprise regulated transcriptioninitiation regions associated with protein translation elongation andthe compositions of this embodiment of the present invention are drawnto novel nucleotide sequences for tissue-independent, tissue-general, orconstitutive plant promoters. By “tissue-independent,” “tissue-general,”or “constitutive” is intended expression in the cells throughout a plantat most times and in most tissues. As with other promoters classified as“constitutive” (e.g., ubiquitin), some variation in absolute levels ofexpression can exist among different tissues or stages.

The present invention thus provides for isolated nucleic acid moleculescomprising a plant nucleotide sequence that directs constitutivetranscription of a linked nucleic acid fragment in a plant cell.Preferably, the nucleotide sequence is obtained from plant genomic DNA.Constitutive promoter sequences may be obtained from other plant speciesby using the constitutive Arabidopsis promoter sequences describedherein as probes to screen for homologous structural genes in otherplants by hybridization under low, moderate or stringent hybridizationconditions. Regions of the constitutive promoter sequences of thepresent invention which are conserved among species could also be usedas PCR primers to amplify a segment from a species other thanArabidopsis, and that segment used as a hybridization probe (the latterapproach permitting higher stringency screening) or in a transcriptionassay to determine promoter activity. Moreover, the constitutivepromoter sequences could be employed to identify structurally relatedsequences in a database using computer algorithms.

These constitutive promoters are capable of driving the expression of acoding sequence in a target cell, particularly in a plant cell. Thepromoter sequences and methods disclosed herein are useful in regulatingconstitutive expression of any heterologous nucleotide sequence in ahost plant in order to vary the phenotype of that plant. These promoterscan be used with combinations of enhancer, upstream elements, and/oractivating sequences from the 5′ flanking regions of plant expressiblestructural genes. Similarly the upstream element can be used incombination with various plant promoter sequences. In one embodiment thepromoter and upstream element are used together to obtain at least10-fold higher expression of an introduced gene in monocot transgenicplants than is obtained with the maize ubiquitin 1 promoter.

In particular, all of the promoters of the invention are useful tomodify the phenotype of a plant. Various changes in the phenotype of atransgenic plant are desirable, i.e., modifying the fatty acidcomposition in a plant, altering the amino acid content of a plant,altering a plant's pathogen defense mechanism, and the like. Theseresults can be achieved by providing expression of heterologous productsor increased expression of endogenous products in plants. Alternatively,the results can be achieved by providing for a reduction of expressionof one or more endogenous products, particularly enzymes or cofactors inthe plant. These changes result in an alteration in the phenotype of thetransformed plant.

I. DEFINITIONS

The term “gene” is used broadly to refer to any segment of nucleic acidassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.For example, “gene” refers to a nucleic acid fragment that expressesmRNA or functional RNA, or encodes a specific protein, and whichincludes regulatory sequences. Genes also include nonexpressed DNAsegments that, for example, form recognition sequences for otherproteins. Genes can be obtained from a variety of sources, includingcloning from a source of interest or synthesizing from known orpredicted sequence information, and may include sequences designed tohave desired parameters.

The term “native” or “wild type” gene refers to a gene that is presentin the genome of an untransformed cell, i.e., a cell not having a knownmutation.

A “marker gene” encodes a selectable or screenable trait.

The term “chimeric gene” refers to any gene that contains 1) DNAsequences, including regulatory and coding sequences, that are not foundtogether in nature, or 2) sequences encoding parts of proteins notnaturally adjoined, or 3) parts of promoters that are not naturallyadjoined. Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orcomprise regulatory sequences and coding sequences derived from the samesource, but arranged in a manner different from that found in nature.

A “transgene” refers to a gene that has been introduced into the genomeby transformation and is stably maintained. Transgenes may include, forexample, genes that are either heterologous or homologous to the genesof a particular plant to be transformed. Additionally, transgenes maycomprise native genes inserted into a non-native organism, or chimericgenes. The term “endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism but that is introduced by genetransfer.

An “oligonucleotide” corresponding to a nucleotide sequence of theinvention, e.g., for use in probing or amplification reactions, may beabout 30 or fewer nucleotides in length (e.g., 9, 12, 15, 18, 20, 21 or24, or any number between 9 and 30). Generally specific primers areupwards of 14 nucleotides in length. For optimum specificity and costeffectiveness, primers of 16 to 24 nucleotides in length may bepreferred. Those skilled in the art are well versed in the design ofprimers for use processes such as PCR. If required, probing can be donewith entire restriction fragments of the gene disclosed herein which maybe 100's or even 1000's of nucleotides in length.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein.

The nucleotide sequences of the invention can be introduced into anyplant. The genes to be introduced can be conveniently used in expressioncassettes for introduction and expression in any plant of interest. Suchexpression cassettes will comprise the transcriptional initiation regionof the invention linked to a nucleotide sequence of interest. Preferredpromoters include constitutive, tissue-specific, developmental-specific,inducible and/or viral promoters. Such an expression cassette isprovided with a plurality of restriction sites for insertion of the geneof interest to be under the transcriptional regulation of the regulatoryregions. The expression cassette may additionally contain selectablemarker genes. The cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, aDNA sequence of interest, and a transcriptional and translationaltermination region functional in plants. The termination region may benative with the transcriptional initiation region, may be native withthe DNA sequence of interest, or may be derived from another source.Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau et al., 1991; Proudfoot, 1991;Sanfacon et al., 1991; Mogen et al., 1990; Munroe et al., 1990; Ballaset al., 1989; Joshi et al., 1987.

“Coding sequence” refers to a DNA or RNA sequence that codes for aspecific amino acid sequence and excludes the non-coding sequences. Itmay constitute an “uninterrupted coding sequence”, i.e., lacking anintron, such as in a cDNA or it may include one or more introns boundedby appropriate splice junctions. An “intron” is a sequence of RNA whichis contained in the primary transcript but which is removed throughcleavage and re-ligation of the RNA within the cell to create the maturemRNA that can be translated into a protein.

The terms “open reading frame” and “ORF” refer to the amino acidsequence encoded between translation initiation and termination codonsof a coding sequence. The terms “initiation codon” and “terminationcodon” refer to a unit of three adjacent nucleotides (‘codon’) in acoding sequence that specifies initiation and chain termination,respectively, of protein synthesis (mRNA translation).

A “functional RNA” refers to an antisense RNA, ribozyme, or other RNAthat is not translated.

The term “RNA transcript” refers to the product resulting from RNApolymerase catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complimentary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” (mRNA) refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a single- or a double-stranded DNA that iscomplementary to and derived from mRNA.

“Regulatory sequences” and “suitable regulatory sequences” each refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences includeenhancers, promoters, translation leader sequences, introns, andpolyadenylation signal sequences. They include natural and syntheticsequences as well as sequences which may be a combination of syntheticand natural sequences. As is noted above, the term “suitable regulatorysequences” is not limited to promoters.

“5′non-coding sequence” refers to a nucleotide sequence located 5′(upstream) to the coding sequence. It is present in the fully processedmRNA upstream of the initiation codon and may affect processing of theprimary transcript to mRNA, mRNA stability or translation efficiency(Turner et al., 1995).

“3′non-coding sequence” refers to nucleotide sequences located 3′(downstream) to a coding sequence and include polyadenylation signalsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al., 1989.

The term “translation leader sequence” refers to that DNA sequenceportion of a gene between the promoter and coding sequence that istranscribed into RNA and is present in the fully processed mRNA upstream(5′) of the translation start codon. The translation leader sequence mayaffect processing of the primary transcript to mRNA, mRNA stability ortranslation efficiency.

The term “mature” protein refers to a post-translationally processedpolypeptide without its signal peptide. “Precursor” protein refers tothe primary product of translation of an mRNA. “Signal peptide” refersto the amino terminal extension of a polypeptide, which is translated inconjunction with the polypeptide forming a precursor peptide and whichis required for its entrance into the secretory pathway. The term“signal sequence” refers to a nucleotide sequence that encodes thesignal peptide.

The term “intracellular localization sequence” refers to a nucleotidesequence that encodes an intracellular targeting signal. An“intracellular targeting signal” is an amino acid sequence that istranslated in conjunction with a protein and directs it to a particularsub-cellular compartment. “Endoplasmic reticulum (ER) stop transitsignal” refers to a carboxy-terminal extension of a polypeptide, whichis translated in conjunction with the polypeptide and causes a proteinthat enters the secretory pathway to be retained in the ER. “ER stoptransit sequence” refers to a nucleotide sequence that encodes the ERtargeting signal. Other intracellular targeting sequences encodetargeting signals active in seeds and/or leaves and vacuolar targetingsignals.

“Promoter” refers to a nucleotide sequence, usually upstream (5′) to itscoding sequence, which controls the expression of the coding sequence byproviding the recognition for RNA polymerase and other factors requiredfor proper transcription. “Promoter” includes a minimal promoter that isa short DNA sequence comprised of a TATA box and other sequences thatserve to specify the site of transcription initiation, to whichregulatory elements are added for control of expression. “Promoter” alsorefers to a nucleotide sequence that includes a minimal promoter plusregulatory elements that is capable of controlling the expression of acoding sequence or functional RNA. This type of promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue specificity of a promoter. It is capable ofoperating in both orientations (normal or flipped), and is capable offunctioning even when moved either upstream or downstream from thepromoter. Both enhancers and other upstream promoter elements bindsequence-specific DNA-binding proteins that mediate their effects.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even be comprised of synthetic DNA segments. A promoter mayalso contain DNA sequences that are involved in the binding of proteinfactors which control the effectiveness of transcription initiation inresponse to physiological or developmental conditions.

The “initiation site” is the position surrounding the first nucleotidethat is part of the transcribed sequence, which is also defined asposition +1. With respect to this site all other sequences of the geneand its controlling regions are numbered. Downstream sequences (i.e.,further protein encoding sequences in the 3′ direction) are denominatedpositive, while upstream sequences (mostly of the controlling regions inthe 5′ direction) are denominated negative.

Promoter elements, particularly a TATA element, that are inactive orthat have greatly reduced promoter activity in the absence of upstreamactivation are referred to as “minimal or core promoters.” In thepresence of a suitable transcription factor, the minimal promoterfunctions to permit transcription. A “minimal or core promoter” thusconsists only of all basal elements needed for transcription initiation,e.g., a TATA box and/or an initiator.

“Constitutive expression” refers to expression using a constitutive orregulated promoter. “Conditional” and “regulated expression” refer toexpression controlled by a regulated promoter.

“Constitutive promoter” refers to a promoter that is able to express theopen reading frame (ORF) that it controls in all or nearly all of theplant tissues during all or nearly all developmental stages of theplant. Each of the transcription-activating elements do not exhibit anabsolute tissue-specificity, but mediate transcriptional activation inmost plant parts at a level of >1% of the level reached in the part ofthe plant in which transcription is most active.

“Regulated promoter” refers to promoters that direct gene expression notconstitutively, but in a temporally- and/or spatially-regulated manner,and includes both tissue-specific and inducible promoters. It includesnatural and synthetic sequences as well as sequences which may be acombination of synthetic and natural sequences. Different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. New promoters of various types useful in plantcells are constantly being discovered, numerous examples may be found inthe compilation by Okamuro et al. (1989). Typical regulated promotersuseful in plants include but are not limited to safener-induciblepromoters, promoters derived from the tetracycline-inducible system,promoters derived from salicylate-inducible systems, promoters derivedfrom alcohol-inducible systems, promoters derived fromglucocorticoid-inducible system, promoters derived frompathogen-inducible systems, and promoters derived fromecdysome-inducible systems.

“Tissue-specific promoter” refers to regulated promoters that are notexpressed in all plant cells but only in one or more cell types inspecific organs (such as leaves or seeds), specific tissues (such asembryo or cotyledon), or specific cell types (such as leaf parenchyma orseed storage cells). These also include promoters that are temporallyregulated, such as in early or late embryogenesis, during fruit ripeningin developing seeds or fruit, in fully differentiated leaf, or at theonset of senescence.

“Inducible promoter” refers to those regulated promoters that can beturned on in one or more cell types by an external stimulus, such as achemical, light, hormone, stress, or a pathogen.

“Operably-linked” refers to the association of nucleic acid sequences onsingle nucleic acid fragment so that the function of one is affected bythe other. For example, a regulatory DNA sequence is said to be“operably linked to” or “associated with” a DNA sequence that codes foran RNA or a polypeptide if the two sequences are situated such that theregulatory DNA sequence affects expression of the coding DNA sequence(i.e., that the coding sequence or functional RNA is under thetranscriptional control of the promoter). Coding sequences can beoperably-linked to regulatory sequences in sense or antisenseorientation.

“Expression” refers to the transcription and/or translation of anendogenous gene, ORF or portion thereof, or a transgene in plants. Forexample, in the case of antisense constructs, expression may refer tothe transcription of the antisense DNA only. In addition, expressionrefers to the transcription and stable accumulation of sense (mRNA) orfunctional RNA. Expression may also refer to the production of protein.

“Specific expression” is the expression of gene products which islimited to one or a few plant tissues (spatial limitation) and/or to oneor a few plant developmental stages (temporal limitation). It isacknowledged that hardly a true specificity exists: promoters seem to bepreferably switch on in some tissues, while in other tissues there canbe no or only little activity. This phenomenon is known as leakyexpression. However, with specific expression in this invention is meantpreferable expression in one or a few plant tissues.

The “expression pattern” of a promoter (with or without enhancer) is thepattern of expression levels which shows where in the plant and in whatdevelopmental stage transcription is initiated by said promoter.Expression patterns of a set of promoters are said to be complementarywhen the expression pattern of one promoter shows little overlap withthe expression pattern of the other promoter. The level of expression ofa promoter can be determined by measuring the ‘steady state’concentration of a standard transcribed reporter mRNA. This measurementis indirect since the concentration of the reporter mRNA is dependentnot only on its synthesis rate, but also on the rate with which the mRNAis degraded. Therefore, the steady state level is the product ofsynthesis rates and degradation rates.

The rate of degradation can however be considered to proceed at a fixedrate when the transcribed sequences are identical, and thus this valuecan serve as a measure of synthesis rates. When promoters are comparedin this way techniques available to those skilled in the art arehybridization S1-RNAse analysis, northern blots and competitive RT-PCR.This list of techniques in no way represents all available techniques,but rather describes commonly used procedures used to analyzetranscription activity and expression levels of mRNA.

The analysis of transcription start points in practically all promotershas revealed that there is usually no single base at which transcriptionstarts, but rather a more or less clustered set of initiation sites,each of which accounts for some start points of the mRNA. Since thisdistribution varies from promoter to promoter the sequences of thereporter mRNA in each of the populations would differ from each other.Since each mRNA species is more or less prone to degradation, no singledegradation rate can be expected for different reporter mRNAs. It hasbeen shown for various eukaryotic promoter sequences that the sequencesurrounding the initiation site (‘initiator’) plays an important role indetermining the level of RNA expression directed by that specificpromoter. This includes also part of the transcribed sequences. Thedirect fusion of promoter to reporter sequences would therefore lead tosuboptimal levels of transcription.

A commonly used procedure to analyze expression patterns and levels isthrough determination of the ‘steady state’ level of proteinaccumulation in a cell. Commonly used candidates for the reporter gene,known to those skilled in the art are β-glucuronidase (GUS),chloramphenicol acetyl transferase (CAT) and proteins with fluorescentproperties, such as green fluorescent protein (GFP) from Aequoravictoria. In principle, however, many more proteins are suitable forthis purpose, provided the protein does not interfere with essentialplant functions. For quantification and determination of localization anumber of tools are suited. Detection systems can readily be created orare available which are based on, e.g., immunochemical, enzymatic,fluorescent detection and quantification. Protein levels can bedetermined in plant tissue extracts or in intact tissue using in situanalysis of protein expression.

Generally, individual transformed lines with one chimeric promoterreporter construct will vary in their levels of expression of thereporter gene. Also frequently observed is the phenomenon that suchtransformants do not express any detectable product (RNA or protein).The variability in expression is commonly ascribed to ‘positioneffects’, although the molecular mechanisms underlying this inactivityare usually not clear.

The term “average expression” is used here as the average level ofexpression found in all lines that do express detectable amounts ofreporter gene, so leaving out of the analysis plants that do not expressany detectable reporter mRNA or protein.

“Root expression level” indicates the expression level found in proteinextracts of complete plant roots. Likewise, leaf, and stem expressionlevels, are determined using whole extracts from leaves and stems. It isacknowledged however, that within each of the plant parts justdescribed, cells with variable functions may exist, in which promoteractivity may vary.

“Non-specific expression” refers to constitutive expression or lowlevel, basal (‘leaky’) expression in nondesired cells or tissues from a‘regulated promoter’.

“Altered levels” refers to the level of expression in transgenicorganisms that differs from that of normal or untransformed organisms.

“Overexpression” refers to the level of expression in transgenic cellsor organisms that exceeds levels of expression in normal oruntransformed (nontransgenic) cells or organisms.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of protein from anendogenous gene or a transgene.

“Co-suppression” and “transwitch” each refer to the production of senseRNA transcripts capable of suppressing the expression of identical orsubstantially similar transgene or endogenous genes (U.S. Pat. No.5,231,020).

“Gene silencing” refers to homology-dependent suppression of viralgenes, transgenes, or endogenous nuclear genes. Gene silencing may betranscriptional, when the suppression is due to decreased transcriptionof the affected genes, or post-transcriptional, when the suppression isdue to increased turnover (degradation) of RNA species homologous to theaffected genes (English et al., 1996). Gene silencing includesvirus-induced gene silencing (Ruiz et al. 1998).

“Silencing suppressor” gene refers to a gene whose expression leads tocounteracting gene silencing and enhanced expression of silenced genes.Silencing suppressor genes may be of plant, non-plant, or viral origin.Examples include, but are not limited to HC-Pro, P1-HC-Pro, and 2bproteins. Other examples include one or more genes in TGMV-B genome.

The terms “heterologous DNA sequence,” “exogenous DNA segment” or“heterologous nucleic acid,” as used herein, each refer to a sequencethat originates from a source foreign to the particular host cell or, iffrom the same source, is modified from its original form. Thus, aheterologous gene in a host cell includes a gene that is endogenous tothe particular host cell but has been modified through, for example, theuse of DNA shuffling. The terms also include non-naturally occurringmultiple copies of a naturally occurring DNA sequence. Thus, the termsrefer to a DNA segment that is foreign or heterologous to the cell, orhomologous to the cell but in a position within the host cell nucleicacid in which the element is not ordinarily found. Exogenous DNAsegments are expressed to yield exogenous polypeptides. A “homologous”DNA sequence is a DNA sequence that is naturally associated with a hostcell into which it is introduced.

“Homologous to” in the context of nucleotide sequence identity refers tothe similarity between the nucleotide sequence of two nucleic acidmolecules or between the amino acid sequences of two protein molecules.Estimates of such homology are provided by either DNA-DNA or DNA-RNAhybridization under conditions of stringency as is well understood bythose skilled in the art (as described in Haines and Higgins (eds.),Nucleic Acid Hybridization, IRL Press, Oxford, U.K.), or by thecomparison of sequence similarity between two nucleic acids or proteins.

The term “substantially similar” refers to nucleotide and amino acidsequences that represent functional and/or structural equivalents ofArabidopsis sequences disclosed herein. For example, altered nucleotidesequences which simply reflect the degeneracy of the genetic code butnonetheless encode amino acid sequences that are identical to aparticular amino acid sequence are substantially similar to theparticular sequences. In addition, amino acid sequences that aresubstantially similar to a particular sequence are those wherein overallamino acid identity is at least 65% or greater to the instant sequences.Modifications that result in equivalent nucleotide or amino acidsequences are well within the routine skill in the art. Moreover, theskilled artisan recognizes that equivalent nucleotide sequencesencompassed by this invention can also be defined by their ability tohybridize, under low, moderate and/or stringent conditions (e.g.,0.1×SSC, 0.1% SDS, 65° C.), with the nucleotide sequences that arewithin the literal scope of the instant claims.

“Target gene” refers to a gene on the replicon that expresses thedesired target coding sequence, functional RNA, or protein. The targetgene is not essential for replicon replication. Additionally, targetgenes may comprise native non-viral genes inserted into a non-nativeorganism, or chimeric genes, and will be under the control of suitableregulatory sequences. Thus, the regulatory sequences in the target genemay come from any source, including the virus. Target genes may includecoding sequences that are either heterologous or homologous to the genesof a particular plant to be transformed. However, target genes do notinclude native viral genes. Typical target genes include, but are notlimited to genes encoding a structural protein, a seed storage protein,a protein that conveys herbicide resistance, and a protein that conveysinsect resistance. Proteins encoded by target genes are known as“foreign proteins”. The expression of a target gene in a plant willtypically produce an altered plant trait.

The term “altered plant trait” means any phenotypic or genotypic changein a transgenic plant relative to the wild-type or non-transgenic planthost.

“Transcription Stop Fragment” refers to nucleotide sequences thatcontain one or more regulatory signals, such as polyadenylation signalsequences, capable of terminating transcription. Examples include the 3′non-regulatory regions of genes encoding nopaline synthase and the smallsubunit of ribulose bisphosphate carboxylase.

“Replication gene” refers to a gene encoding a viral replicationprotein. In addition to the ORF of the replication protein, thereplication gene may also contain other overlapping or non-overlappingORF(s), as are found in viral sequences in nature. While not essentialfor replication, these additional ORFs may enhance replication and/orviral DNA accumulation. Examples of such additional ORFs are AC3 and AL3in ACMV and TGMV geminiviruses, respectively.

“Chimeric trans-acting replication gene” refers either to a replicationgene in which the coding sequence of a replication protein is under thecontrol of a regulated plant promoter other than that in the nativeviral replication gene, or a modified native viral replication gene, forexample, in which a site specific sequence(s) is inserted in the 5′transcribed but untranslated region. Such chimeric genes also includeinsertion of the known sites of replication protein binding between thepromoter and the transcription start site that attenuate transcriptionof viral replication protein gene.

“Chromosomally-integrated” refers to the integration of a foreign geneor DNA construct into the host DNA by covalent bonds. Where genes arenot “chromosomally integrated” they may be “transiently expressed.”Transient expression of a gene refers to the expression of a gene thatis not integrated into the host chromosome but functions independently,either as part of an autonomously replicating plasmid or expressioncassette, for example, or as part of another biological system such as avirus.

“Production tissue” refers to mature, harvestable tissue consisting ofnon-dividing, terminally-differentiated cells. It excludes young,growing tissue consisting of germline, meristematic, andnot-fully-differentiated cells.

“Germline cells” refer to cells that are destined to be gametes andwhose genetic material is heritable.

“Trans-activation” refers to switching on of gene expression or repliconreplication by the expression of another (regulatory) gene in trans.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, resulting in genetically stableinheritance. Host cells containing the transformed nucleic acidfragments are referred to as “transgenic” cells, and organismscomprising transgenic cells are referred to as “transgenic organisms”.Examples of methods of transformation of plants and plant cells includeAgrobacterium-mediated transformation (De Blaere et al., 1987) andparticle bombardment technology (Klein et al. 1987; U.S. Pat. No.4,945,050). Whole plants may be regenerated from transgenic cells bymethods well known to the skilled artisan (see, for example, Fromm etal., 1990).

“Transformed,” “transgenic,” and “recombinant” refer to a host organismsuch as a bacterium or a plant into which a heterologous nucleic acidmolecule has been introduced. The nucleic acid molecule can be stablyintegrated into the genome generally known in the art and are disclosedin Sambrook et al., 1989. See also Innis et al., 1995 and Gelfand, 1995;and Innis and Gelfand, 1999. Known methods of PCR include, but are notlimited to, methods using paired primers, nested primers, singlespecific primers, degenerate primers, gene-specific primers,vector-specific primers, partially mismatched primers, and the like. Forexample, “transformed,” “transformant,” and “transgenic” plants or callihave been through the transformation process and contain a foreign geneintegrated into their chromosome. The term “untransformed” refers tonormal plants that have not been through the transformation process.

“Transiently transformed” refers to cells in which transgenes andforeign DNA have been introduced (for example, by such methods asAgrobacterium-mediated transformation or biolistic bombardment), but notselected for stable maintenance.

“Stably transformed” refers to cells that have been selected andregenerated on a selection media following transformation.

“Transient expression” refers to expression in cells in which a virus ora transgene is introduced by viral infection or by such methods asAgrobacterium-mediated transformation, electroporation, or biolisticbombardment, but not selected for its stable maintenance.

“Genetically stable” and “heritable” refer to chromosomally-integratedgenetic elements that are stably maintained in the plant and stablyinherited by progeny through successive generations.

“Primary transformant” and “TO generation” refer to transgenic plantsthat are of the same genetic generation as the tissue which wasinitially transformed (i.e., not having gone through meiosis andfertilization since transformation).

“Secondary transformants” and the “T1, T2, T3, etc. generations” referto transgenic plants derived from primary transformants through one ormore meiotic and fertilization cycles. They may be derived byself-fertilization of primary or secondary transformants or crosses ofprimary or secondary transformants with other transformed oruntransformed plants.

“Wild-type” refers to a virus or organism found in nature without anyknown mutation.

“Genome” refers to the complete genetic material of an organism.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base which is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides which have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., 1991; Ohtsuka et al., 1985;Rossolini et al. 1994). A “nucleic acid fragment” is a fraction of agiven nucleic acid molecule. In higher plants, deoxyribonucleic acid(DNA) is the genetic material while ribonucleic acid (RNA) is involvedin the transfer of information contained within DNA into proteins. Theterm “nucleotide sequence” refers to a polymer of DNA or RNA which canbe single- or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases capable of incorporation intoDNA or RNA polymers. The terms “nucleic acid” or “nucleic acid sequence”may also be used interchangeably with gene, cDNA, DNA and RNA encoded bya gene.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. In the context of the present invention,an “isolated” or “purified” DNA molecule or an “isolated” or “purified”polypeptide is a DNA molecule or polypeptide that, by the hand of man,exists apart from its native environment and is therefore not a productof nature. An isolated DNA molecule or polypeptide may exist in apurified form or may exist in a non-native environment such as, forexample, a transgenic host cell. For example, an “isolated” or“purified” nucleic acid molecule or protein, or biologically activeportion thereof, is substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized. Preferably, an “isolated” nucleic acid is free of sequences(preferably protein encoding sequences) that naturally flank the nucleicacid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleicacid molecule in genomic DNA of the cell from which the nucleic acid isderived. A protein that is substantially free of cellular materialincludes preparations of protein or polypeptide having less than about30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When theprotein of the invention, or biologically active portion thereof, isrecombinantly produced, preferably culture medium represents less thanabout 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors ornon-protein of interest chemicals.

The nucleotide sequences of the invention include both the naturallyoccurring sequences as well as mutant (variant) forms. Such variantswill continue to possess the desired activity, i.e., either promoteractivity or the activity of the product encoded by the open readingframe of the non-variant nucleotide sequence.

Thus, by “variants” is intended substantially similar sequences. Fornucleotide sequences comprising an open reading frame, variants includethose sequences that, because of the degeneracy of the genetic code,encode the identical amino acid sequence of the native protein.Naturally occurring allelic variants such as these can be identifiedwith the use of well-known molecular biology techniques, as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques. Variant nucleotide sequences also include syntheticallyderived nucleotide sequences, such as those generated, for example, byusing site-directed mutagenesis and for open reading frames, encode thenative protein, as well as those that encode a polypeptide having aminoacid substitutions relative to the native protein. Generally, nucleotidesequence variants of the invention will have at least 40, 50, 60, to70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%,generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99%nucleotide sequence identity to the native (wild type or endogenous)nucleotide sequence.

“Conservatively modified variations” of a particular nucleic acidsequence refers to those nucleic acid sequences that encode identical oressentially identical amino acid sequences, or where the nucleic acidsequence does not encode an amino acid sequence, to essentiallyidentical sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenpolypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGGall encode the amino acid arginine. Thus, at every position where anarginine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded protein.Such nucleic acid variations are “silent variations” which are onespecies of “conservatively modified variations.” Every nucleic acidsequence described herein which encodes a polypeptide also describesevery possible silent variation, except where otherwise noted. One ofskill will recognize that each codon in a nucleic acid (except ATG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

The nucleic acid molecules of the invention can be “optimized” forenhanced expression in plants of interest. See, for example, EPA 035472;WO 91/16432; Perlak et al., 1991; and Murray et al., 1989. In thismanner, the open reading frames in genes or gene fragments can besynthesized utilizing plant-preferred codons. See, for example, Campbelland Gowri, 1990 for a discussion of host-preferred codon usage. Thus,the nucleotide sequences can be optimized for expression in any plant.It is recognized that all or any part of the gene sequence may beoptimized or synthetic. That is, synthetic or partially optimizedsequences may also be used. Variant nucleotide sequences and proteinsalso encompass sequences and protein derived from a mutagenic andrecombinogenic procedure such as DNA shuffling. With such a procedure,one or more different coding sequences can be manipulated to create anew polypeptide possessing the desired properties. In this manner,libraries of recombinant polynucleotides are generated from a populationof related sequence polynucleotides comprising sequence regions thathave substantial sequence identity and can be homologously recombined invitro or in vivo. Strategies for such DNA shuffling are known in theart. See, for example, Stemmer, 1994; Stemmer, 1994; Crameri et al.,1997; Moore et al., 1997; Zhang et al., 1997; Crameri et al., 1998; andU.S. Pat. Nos. 5,605,793 and 5,837,458.

By “variant” polypeptide is intended a polypeptide derived from thenative protein by deletion (so-called truncation) or addition of one ormore amino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Such variants may resultfrom, for example, genetic polymorphism or from human manipulation.Methods for such manipulations are generally known in the art.

Thus, the polypeptides may be altered in various ways including aminoacid substitutions, deletions, truncations, and insertions. Methods forsuch manipulations are generally known in the art. For example, aminoacid sequence variants of the polypeptides can be prepared by mutationsin the DNA. Methods for mutagenesis and nucleotide sequence alterationsare well known in the art. See, for example, Kunkel, 1985; Kunkel etal., 1987; U.S. Pat. No. 4,873,192; Walker and Gaastra, 1983 and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978).Conservative substitutions, such as exchanging one amino acid withanother having similar properties, are preferred.

Individual substitutions deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids(typically less than 5%, more typically less than 1%) in an encodedsequence are “conservatively modified variations,” where the alterationsresult in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following five groupseach contain amino acids that are conservative substitutions for oneanother: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L),Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan(W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine I,Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E),Asparagine (N), Glutamine (Q). See also, Creighton, 1984. In addition,individual substitutions, deletions or additions which alter, add ordelete a single amino acid or a small percentage of amino acids in anencoded sequence are also “conservatively modified variations.”

“Expression cassette” as used herein means a DNA sequence capable ofdirecting expression of a particular nucleotide sequence in anappropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The coding region usually codesfor a protein of interest but may also code for a functional RNA ofinterest, for example antisense RNA or a nontranslated RNA, in the senseor antisense direction. The expression cassette comprising thenucleotide sequence of interest may be chimeric, meaning that at leastone of its components is heterologous with respect to at least one ofits other components. The expression cassette may also be one which isnaturally occurring but has been obtained in a recombinant form usefulfor heterologous expression. The expression of the nucleotide sequencein the expression cassette may be under the control of a constitutivepromoter or of an inducible promoter which initiates transcription onlywhen the host cell is exposed to some particular external stimulus. Inthe case of a multicellular organism, the promoter can also be specificto a particular tissue or organ or stage of development.

“Vector” is defined to include, inter alia, any plasmid, cosmid, phageor Agrobacterium binary vector in double or single stranded linear orcircular form which may or may not be self transmissible or mobilizable,and which can transform a prokaryotic or eukaryotic host either byintegration into the cellular genome or exist extrachromosomally (e.g.autonomous replicating plasmid with an origin of replication).

Specifically included are shuttle vectors by which is meant a DNAvehicle capable, naturally or by design, of replication in two differenthost organisms, which may be selected from actinomycetes and relatedspecies, bacteria and eukaryotic (e.g. higher plant, mammalian, yeast orfungal cells).

Preferably the nucleic acid in the vector is under the control of, andoperably linked to, an appropriate promoter or other regulatory elementsfor transcription in a host cell such as a microbial, e.g. bacterial, orplant cell. The vector may be a bi-functional expression vector whichfunctions in multiple hosts. In the case of genomic DNA, this maycontain its own promoter or other regulatory elements and in the case ofcDNA this may be under the control of an appropriate promoter or otherregulatory elements for expression in the host cell.

“Cloning vectors” typically contain one or a small number of restrictionendonuclease recognition sites at which foreign DNA sequences can beinserted in a determinable fashion without loss of essential biologicalfunction of the vector, as well as a marker gene that is suitable foruse in the identification and selection of cells transformed with thecloning vector. Marker genes typically include genes that providetetracycline resistance, hygromycin resistance or ampicillin resistance.

A “transgenic plant” is a plant having one or more plant cells thatcontain an expression vector.

“Plant tissue” includes differentiated and undifferentiated tissues orplants, including but not limited to roots, stems, shoots, leaves,pollen, seeds, tumor tissue and various forms of cells and culture suchas single cells, protoplast, embryos, and callus tissue. The planttissue may be in plants or in organ, tissue or cell culture.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm. Preferred,non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller, 1988; the local homology algorithm of Smith et al.1981; the homology alignment algorithm of Needleman and Wunsch 1970; thesearch-for-similarity-method of Pearson and Lipman 1988; the algorithmof Karlin and Altschul, 1990, modified as in Karlin and Altschul, 1993.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.1988; Higgins et al. 1989; Corpet et al. 1988; Huang et al. 1992; andPearson et al. 1994. The ALIGN program is based on the algorithm ofMyers and Miller, supra. The BLAST programs of Altschul et al., 1990,are based on the algorithm of Karlin and Altschul supra.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., 1990). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when the cumulative alignment score falls off bythe quantity X from its maximum achieved value, the cumulative scoregoes to zero or below due to the accumulation of one or morenegative-scoring residue alignments, or the end of either sequence isreached.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993). One measure ofsimilarity provided by the BLAST algorithm is the smallest sumprobability (P(N)), which provides an indication of the probability bywhich a match between two nucleotide or amino acid sequences would occurby chance. For example, a test nucleic acid sequence is consideredsimilar to a reference sequence if the smallest sum probability in acomparison of the test nucleic acid sequence to the reference nucleicacid sequence is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al. 1997.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al., supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.BLASTN for nucleotide sequences, BLASTX for proteins) can be used. TheBLASTN program (for nucleotide sequences) uses as defaults a wordlength(W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989). Alignmentmay also be performed manually by inspection.

For purposes of the present invention, comparison of nucleotidesequences for determination of percent sequence identity to the promotersequences disclosed herein is preferably made using the BlastN program(version 1.4.7 or later) with its default parameters or any equivalentprogram. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide or amino acid residue matches and anidentical percent sequence identity when compared to the correspondingalignment generated by the preferred program.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%,91%, 92%, 93%, or 94%, and most preferably at least 95%, 96%, 97%, 98%,or 99% sequence identity, compared to a reference sequence using one ofthe alignment programs described using standard parameters. One of skillin the art will recognize that these values can be appropriatelyadjusted to determine corresponding identity of proteins encoded by twonucleotide sequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning, and the like. Substantialidentity of amino acid sequences for these purposes normally meanssequence identity of at least 70%, more preferably at least 80%, 90%,and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions(see below). Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. However, stringentconditions encompass temperatures in the range of about 1° C. to about20° C., depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid is immunologically crossreactive with the polypeptide encoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%,92%, 93%, or 94%, or even more preferably, 95%, 96%, 97%, 98% or 99%,sequence identity to the reference sequence over a specified comparisonwindow. Preferably, optimal alignment is conducted using the homologyalignment algorithm of Needleman and Wunsch (1970). An indication thattwo peptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

As noted above, another indication that two nucleic acid sequences aresubstantially identical is that the two molecules hybridize to eachother under stringent conditions. The phrase “hybridizing specificallyto” refers to the binding, duplexing, or hybridizing of a molecule onlyto a particular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridization are sequence dependent, andare different under different environmental parameters. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Specificity istypically the function of post-hybridization washes, the criticalfactors being the ionic strength and temperature of the final washsolution. For DNA-DNA hybrids, the T_(m) can be approximated from theequation of Meinkoth and Wahl, 1984; T_(m) 81.5° C.+16.6 (log M)+0.41 (%GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations,% GC is the percentage of guanosine and cytosine nucleotides in the DNA,% form is the percentage of formamide in the hybridization solution, andL is the length of the hybrid in base pairs. T_(m) is reduced by about1° C. for each 1% of mismatching; thus, T_(m), hybridization, and/orwash conditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point I forthe specific sequence and its complement at a defined ionic strength andpH. However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point I;moderately stringent conditions can utilize a hybridization and/or washat 6, 7, 8, 9, or 10° C. lower than the thermal melting point I; lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the thermal melting point 1. Using theequation, hybridization and wash compositions, and desired T, those ofordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a T of less than 45° C.(aqueous solution) or 32° C. (formamide solution), it is preferred toincrease the SSC concentration so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found inTijssen, 1993. Generally, highly stringent hybridization and washconditions are selected to be about 5° C. lower than the thermal meltingpoint T_(m) for the specific sequence at a defined ionic strength andpH.

An example of highly stringent wash conditions is 0.15 M NaCl at 72° C.for about 15 minutes. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook, infra, for adescription of SSC buffer). Often, a high stringency wash is preceded bya low stringency wash to remove background probe signal. An examplemedium stringency wash for a duplex of, e.g., more than 100 nucleotides,is 1×SSC at 45° C. for 15 minutes. An example low stringency wash for aduplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15minutes. For short probes (e.g., about 10 to 50 nucleotides), stringentconditions typically involve salt concentrations of less than about 1.5M, more preferably about 0.01 to 1.0 M, Na ion concentration (or othersalts) at pH 7.0 to 8.3, and the temperature is typically at least about30° C. and at least about 60° C. for long robes (e.g., >50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleic acids that do not hybridize to each other understringent conditions are still substantially identical if the proteinsthat they encode are substantially identical. This occurs, e.g., when acopy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Very stringent conditions are selected to be equal to the T_(m) for aparticular probe. An example of stringent conditions for hybridizationof complementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamide,e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1 MNaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1.times.to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C.Exemplary moderate stringency conditions include hybridization in 40 to45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSCat 55 to 60° C.

The following are examples of sets of hybridization/wash conditions thatmay be used to clone orthologous nucleotide sequences that aresubstantially identical to reference nucleotide sequences of the presentinvention: a reference nucleotide sequence preferably hybridizes to thereference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C.,more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirablystill in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C. with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,0.1% SDS at 65° C.

“DNA shuffling” is a method to introduce mutations or rearrangements,preferably randomly, in a DNA molecule or to generate exchanges of DNAsequences between two or more DNA molecules, preferably randomly. TheDNA molecule resulting from DNA shuffling is a shuffled DNA moleculethat is a non-naturally occurring DNA molecule derived from at least onetemplate DNA molecule. The shuffled DNA preferably encodes a variantpolypeptide modified with respect to the polypeptide encoded by thetemplate DNA, and may have an altered biological activity with respectto the polypeptide encoded by the template DNA.

“Recombinant DNA molecule” is a combination of DNA sequences that arejoined together using recombinant DNA technology and procedures used tojoin together DNA sequences as described, for example, in Sambrook etal., 1989.

The word “plant” refers to any plant, particularly to seed plant, and“plant cell” is a structural and physiological unit of the plant, whichcomprises a cell wall but may also refer to a protoplast. The plant cellmay be in form of an isolated single cell or a cultured cell, or as apart of higher organized unit such as, for example, a plant tissue, or aplant organ.

“Significant increase” is an increase that is larger than the margin oferror inherent in the measurement technique, preferably an increase byabout 2-fold or greater.

“Significantly less” means that the decrease is larger than the marginof error inherent in the measurement technique, preferably a decrease byabout 2-fold or greater.

II. DNA SEQUENCES FOR TRANSFORMATION

Virtually any DNA composition may be used for delivery to recipientplant cells, e.g., monocotyledonous cells, to ultimately produce fertiletransgenic plants in accordance with the present invention. For example,DNA segments in the form of vectors and plasmids, or linear DNAfragments, in some instances containing only the DNA element to beexpressed in the plant, and the like, may be employed. The constructionof vectors which may be employed in conjunction with the presentinvention will be known to those of skill of the art in light of thepresent disclosure (see, e.g., Sambrook et al., 1989; Gelvin et al.,1990).

Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes), BACs(bacterial artificial chromosomes) and DNA segments for use intransforming such cells will, of course, generally comprise the cDNA,gene or genes which one desires to introduce into the cells. These DNAconstructs can further include structures such as promoters, enhancers,polylinkers, or even regulatory genes as desired. The DNA segment orgene chosen for cellular introduction will often encode a protein whichwill be expressed in the resultant recombinant cells, such as willresult in a screenable or selectable trait and/or which will impart animproved phenotype to the regenerated plant. However, this may notalways be the case, and the present invention also encompassestransgenic plants incorporating non-expressed transgenes.

In certain embodiments, it is contemplated that one may wish to employreplication-competent viral vectors in monocot transformation. Suchvectors include, for example, wheat dwarf virus (WDV) “shuttle” vectors,such as pW1-11 and PW1-GUS (Ugaki et al., 1991). These vectors arecapable of autonomous replication in maize cells as well as E. coli, andas such may provide increased sensitivity for detecting DNA delivered totransgenic cells. A replicating vector may also be useful for deliveryof genes flanked by DNA sequences from transposable elements such as Ac,Ds, or Mu. It has been proposed (Laufs et al., 1990) that transpositionof these elements within the maize genome requires DNA replication. Itis also contemplated that transposable elements would be useful forintroducing DNA fragments lacking elements necessary for selection andmaintenance of the plasmid vector in bacteria, e.g., antibioticresistance genes and origins of DNA replication. It is also proposedthat use of a transposable element such as Ac, Ds, or Mu would activelypromote integration of the desired DNA and hence increase the frequencyof stably transformed cells. The use of a transposable element such asAc, Ds, or Mu may actively promote integration of the DNA of interestand hence increase the frequency of stably transformed cells.Transposable elements may be useful to allow separation of genes ofinterest from elements necessary for selection and maintenance of aplasmid vector in bacteria or selection of a transformant. By use of atransposable element, desirable and undesirable DNA sequences may betransposed apart from each other in the genome, such that throughgenetic segregation in progeny, one may identify plants with either thedesirable or the undesirable DNA sequences.

DNA useful for introduction into plant cells includes that which hasbeen derived or isolated from any source, that may be subsequentlycharacterized as to structure, size and/or function, chemically altered,and later introduced into plants. An example of DNA “derived” from asource, would be a DNA sequence that is identified as a useful fragmentwithin a given organism, and which is then chemically synthesized inessentially pure form. An example of such DNA “isolated” from a sourcewould be a useful DNA sequence that is excised or removed from saidsource by chemical means, e.g., by the use of restriction endonucleases,so that it can be further manipulated, e.g., amplified, for use in theinvention, by the methodology of genetic engineering. Such DNA iscommonly referred to as “recombinant DNA.”

Therefore useful DNA includes completely synthetic DNA, semi-syntheticDNA, DNA isolated from biological sources, and DNA derived fromintroduced RNA. Generally, the introduced DNA is not originally residentin the plant genotype which is the recipient of the DNA, but it iswithin the scope of the invention to isolate a gene from a given plantgenotype, and to subsequently introduce multiple copies of the gene intothe same genotype, e.g., to enhance production of a given gene productsuch as a storage protein or a protein that confers tolerance orresistance to water deficit.

The introduced DNA includes but is not limited to, DNA from plant genes,and non-plant genes such as those from bacteria, yeasts, animals orviruses. The introduced DNA can include modified genes, portions ofgenes, or chimeric genes, including genes from the same or differentmaize genotype. The term “chimeric gene” or “chimeric DNA” is defined asa gene or DNA sequence or segment comprising at least two DNA sequencesor segments from species which do not combine DNA under naturalconditions, or which DNA sequences or segments are positioned or linkedin a manner which does not normally occur in the native genome ofuntransformed plant.

The introduced DNA used for transformation herein may be circular orlinear, double-stranded or single-stranded. Generally, the DNA is in theform of chimeric DNA, such as plasmid DNA, that can also contain codingregions flanked by regulatory sequences which promote the expression ofthe recombinant DNA present in the resultant plant. For example, the DNAmay itself comprise or consist of a promoter that is active in a plantwhich is derived from a source other than that plant, or may utilize apromoter already present in a plant genotype that is the transformationtarget.

Generally, the introduced DNA will be relatively small, i.e., less thanabout 30 kb to minimize any susceptibility to physical, chemical, orenzymatic degradation which is known to increase as the size of the DNAincreases. As noted above, the number of proteins, RNA transcripts ormixtures thereof which is introduced into the plant genome is preferablypreselected and defined, e.g., from one to about 5-10 such products ofthe introduced DNA may be formed.

Two principal methods for the control of expression are known, viz.:overexpression and underexpression. Overexpression can be achieved byinsertion of one or more than one extra copy of the selected gene. Itis, however, not unknown for plants or their progeny, originallytransformed with one or more than one extra copy of a nucleotidesequence, to exhibit the effects of underexpression as well asoverexpression. For underexpression there are two principle methodswhich are commonly referred to in the art as “antisense downregulation”and “sense downregulation” (sense downregulation is also referred to as“cosuppression”). Generically these processes are referred to as “genesilencing”. Both of these methods lead to an inhibition of expression ofthe target gene.

Obtaining sufficient levels of transgene expression in the appropriateplant tissues is an important aspect in the production of geneticallyengineered crops. Expression of heterologous DNA sequences in a planthost is dependent upon the presence of an operably linked promoter thatis functional within the plant host. Choice of the promoter sequencewill determine when and where within the organism the heterologous DNAsequence is expressed.

Furthermore, it is contemplated that promoters combining elements frommore than one promoter may be useful. For example, U.S. Pat. No.5,491,288 discloses combining a Cauliflower Mosaic Virus promoter with ahistone promoter. Thus, the elements from the promoters disclosed hereinmay be combined with elements from other promoters.

Promoters which are useful for plant transgene expression include thosethat are inducible, viral, synthetic, constitutive (Odell et al., 1985),temporally regulated, spatially regulated, tissue-specific, andspatio-temporally regulated.

Where expression in specific tissues or organs is desired,tissue-specific promoters may be used. In contrast, where geneexpression in response to a stimulus is desired, inducible promoters arethe regulatory elements of choice. Where continuous expression isdesired throughout the cells of a plant, constitutive promoters areutilized. Additional regulatory sequences upstream and/or downstreamfrom the core promoter sequence may be included in expression constructsof transformation vectors to bring about varying levels of expression ofheterologous nucleotide sequences in a transgenic plant.

A. Transcription Regulatory Sequences

1. Promoters

The choice of promoter will vary depending on the temporal and spatialrequirements for expression, and also depending on the target species.In some cases, expression in multiple tissues is desirable. While inothers, tissue-specific, e.g., leaf-specific, expression is desirable.Although many promoters from dicotyledons have been shown to beoperational in monocotyledons and vice versa, ideally dicotyledonouspromoters are selected for expression in dicotyledons, andmonocotyledonous promoters for expression in monocotyledons. However,there is no restriction to the provenance of selected promoters; it issufficient that they are operational in driving the expression of thenucleotide sequences in the desired cell.

These promoters include, but are not limited to, constitutive,inducible, temporally regulated, developmentally regulated,spatially-regulated, chemically regulated, stress-responsive,tissue-specific, viral and synthetic promoters. Promoter sequences areknown to be strong or weak. A strong promoter provides for a high levelof gene expression, whereas a weak promoter provides for a very lowlevel of gene expression. An inducible promoter is a promoter thatprovides for the turning on and off of gene expression in response to anexogenously added agent, or to an environmental or developmentalstimulus. A bacterial promoter such as the P_(tac) promoter can beinduced to varying levels of gene expression depending on the level ofisothiopropylgalactoside added to the transformed bacterial cells. Anisolated promoter sequence that is a strong promoter for heterologousnucleic acid is advantageous because it provides for a sufficient levelof gene expression to allow for easy detection and selection oftransformed cells and provides for a high level of gene expression whendesired.

Within a plant promoter region there are several domains that arenecessary for full function of the promoter. The first of these domainslies immediately upstream of the structural gene and forms the “corepromoter region” containing consensus sequences, normally 70 base pairsimmediately upstream of the gene. The core promoter region contains thecharacteristic CAAT and TATA boxes plus surrounding sequences, andrepresents a transcription initiation sequence that defines thetranscription start point for the structural gene.

The presence of the core promoter region defines a sequence as being apromoter: if the region is absent, the promoter is non-functional.Furthermore, the core promoter region is insufficient to provide fullpromoter activity. A series of regulatory sequences upstream of the coreconstitute the remainder of the promoter. The regulatory sequencesdetermine expression level, the spatial and temporal pattern ofexpression and, for an important subset of promoters, expression underinductive conditions (regulation by external factors such as light,temperature, chemicals, hormones).

A range of naturally-occurring promoters are known to be operative inplants and have been used to drive the expression of heterologous (bothforeign and endogenous) genes in plants: for example, the constitutive35S cauliflower mosaic virus (CaMV) promoter, the ripening-enhancedtomato polygalacturonase promoter (Bird et al., 1988), the E8 promoter(Diekman & Fischer, 1988) and the fruit specific 2A1 promoter (Pear etal., 1989) and many others, e.g., U2 and U5 snRNA promoters from maize,the promoter from alcohol dehydrogenase, the Z4 promoter from a geneencoding the Z4 22 kD zein protein, the Z10 promoter from a geneencoding a 10 kD zein protein, a Z27 promoter from a gene encoding a 27kD zein protein, the A20 promoter from the gene encoding a 19 kD-zeinprotein, inducible promoters, such as the light inducible promoterderived from the pea rbcS gene and the actin promoter from rice, e.g.,the actin 2 promoter (WO 00/70067); seed specific promoters, such as thephaseolin promoter from beans, may also be used. The nucleotidesequences of this invention can also be expressed under the regulationof promoters that are chemically regulated. This enables the nucleicacid sequence or encoded polypeptide to be synthesized only when thecrop plants are treated with the inducing chemicals. Chemical inductionof gene expression is detailed in EP 0 332 104 (to Ciba-Geigy) and U.S.Pat. No. 5,614,395. A preferred promoter for chemical induction is thetobacco PR-1a promoter.

Examples of some constitutive promoters which have been describedinclude the rice actin 1 (Wang et al., 1992; U.S. Pat. No. 5,641,876),CaMV 35S (Odell et al., 1985), CaMV 19S (Lawton et al., 1987), nos, Adh,sucrose synthase; and the ubiquitin promoters.

Examples of tissue specific promoters which have been described includethe lectin (Vodkin, 1983; Lindstrom et al., 1990) corn alcoholdehydrogenase 1 (Vogel et al., 1989; Dennis et al., 1984), corn lightharvesting complex (Simpson, 1986; Bansal et al., 1992), corn heat shockprotein (Odell et al., 1985), pea small subunit RuBP carboxylase(Poulsen et al., 1986), Ti plasmid mannopine synthase (Langridge et al.,1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petuniachalcone isomerase (vanTunen et al., 1988), bean glycine rich protein 1(Keller et al., 1989), truncated CaMV 35S (Odell et al., 1985), potatopatatin (Wenzler et al., 1989), root cell (Yamamoto et al., 1990), maizezein (Reina et al., 1990; Kriz et al., 1987; Wandelt et al., 1989;Langridge et al., 1983; Reina et al., 1990), globulin-1 (Belanger etal., 1991), α-tubulin, cab (Sullivan et al., 1989), PEPCase (Hudspeth &Grula, 1989), R gene complex-associated promoters (Chandler et al.,1989), histone, and chalcone synthase promoters (Franken et al., 1991).Tissue specific enhancers are described in Fromm et al. (1989).

Inducible promoters that have been described include the ABA- andturgor-inducible promoters, the promoter of the auxin-binding proteingene (Schwob et al., 1993), the UDP glucose flavonoidglycosyl-transferase gene promoter (Ralston et al., 1988), the MPIproteinase inhibitor promoter (Cordero et al., 1994), and theglyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al.,1995; Quigley et al., 1989; Martinez et al., 1989).

Several other tissue-specific regulated genes and/or promoters have beenreported in plants. These include genes encoding the seed storageproteins (such as napin, cruciferin, beta-conglycinin, and phaseolin)zein or oil body proteins (such as oleosin), or genes involved in fattyacid biosynthesis (including acyl carrier protein, stearoyl-ACPdesaturase. And fatty acid desaturases (fad 2-1)), and other genesexpressed during embryo development (such as Bce4, see, for example, EP255378 and Kridl et al., 1991). Particularly useful for seed-specificexpression is the pea vicilin promoter (Czako et al., 1992). (See alsoU.S. Pat. No. 5,625,136, herein incorporated by reference). Other usefulpromoters for expression in mature leaves are those that are switched onat the onset of senescence, such as the SAG promoter from Arabidopsis(Gan et al., 1995).

A class of fruit-specific promoters expressed at or during antithesisthrough fruit development, at least until the beginning of ripening, isdiscussed in U.S. Pat. No. 4,943,674. cDNA clones that arepreferentially expressed in cotton fiber have been isolated (John etal., 1992). cDNA clones from tomato displaying differential expressionduring fruit development have been isolated and characterized (Manssonet al., 1985, Slater et al., 1985). The promoter for polygalacturonasegene is active in fruit ripening. The polygalacturonase gene isdescribed in U.S. Pat. No. 4,535,060, U.S. Pat. No. 4,769,061, U.S. Pat.No. 4,801,590, and U.S. Pat. No. 5,107,065, which disclosures areincorporated herein by reference.

Other examples of tissue-specific promoters include those that directexpression in leaf cells following damage to the leaf (for example, fromchewing insects), in tubers (for example, patatin gene promoter), and infiber cells (an example of a developmentally-regulated fiber cellprotein is E6 (John et al., 1992). The E6 gene is most active in fiber,although low levels of transcripts are found in leaf, ovule and flower.

The tissue-specificity of some “tissue-specific” promoters may not beabsolute and may be tested by one skilled in the art using thediphtheria toxin sequence. One can also achieve tissue-specificexpression with “leaky” expression by a combination of differenttissue-specific promoters (Beals et al., 1997). Other tissue-specificpromoters can be isolated by one skilled in the art (see U.S. Pat. No.5,589,379). Several inducible promoters (“gene switches”) have beenreported. Many are described in the review by Gatz (1996) and Gatz(1997). These include tetracycline repressor system, Lac repressorsystem, copper-inducible systems, salicylate-inducible systems (such asthe PR1a system), glucocorticoid—(Aoyama et al., 1997) andecdysome-inducible systems. Also included are the benzenesulphonamide—(U.S. Pat. No. 5,364,780) and alcohol—(WO 97/06269 and WO97/06268) inducible systems and glutathione S-transferase promoters.Other studies have focused on genes inducibly regulated in response toenvironmental stress or stimuli such as increased salinity. Drought,pathogen and wounding. (Graham et al., 1985; Graham et al., 1985, Smithet al., 1986). Accumulation of metallocarboxypeptidase-inhibitor proteinhas been reported in leaves of wounded potato plants (Graham et al.,1981). Other plant genes have been reported to be induced methyljasmonate, elicitors, heat-shock, anaerobic stress, or herbicidesafeners.

Regulated expression of the chimeric transacting viral replicationprotein can be further regulated by other genetic strategies. Forexample, Cre-mediated gene activation as described by Odell et al. 1990.Thus, a DNA fragment containing 3′ regulatory sequence bound by loxsites between the promoter and the replication protein coding sequencethat blocks the expression of a chimeric replication gene from thepromoter can be removed by Cre-mediated excision and result in theexpression of the trans-acting replication gene. In this case, thechimeric Cre gene, the chimeric trans-acting replication gene, or bothcan be under the control of tissue- and developmental-specific orinducible promoters. An alternate genetic strategy is the use of tRNAsuppressor gene. For example, the regulated expression of a tRNAsuppressor gene can conditionally control expression of a trans-actingreplication protein coding sequence containing an appropriatetermination codon as described by Ulmasov et al. 1997. Again, either thechimeric tRNA suppressor gene, the chimeric transacting replicationgene, or both can be under the control of tissue- anddevelopmental-specific or inducible promoters.

Frequently it is desirable to have continuous or inducible expression ofa DNA sequence throughout the cells of an organism in atissue-independent manner. For example, increased resistance of a plantto infection by soil- and airborne-pathogens might be accomplished bygenetic manipulation of the plant's genome to comprise a continuouspromoter operably linked to a heterologous pathogen-resistance gene suchthat pathogen-resistance proteins are continuously expressed throughoutthe plant's tissues.

Alternatively, it might be desirable to inhibit expression of a nativeDNA sequence within a plant's tissues to achieve a desired phenotype. Inthis case, such inhibition might be accomplished with transformation ofthe plant to comprise a constitutive, tissue-independent promoteroperably linked to an antisense nucleotide sequence, such thatconstitutive expression of the antisense sequence produces an RNAtranscript that interferes with translation of the mRNA of the nativeDNA sequence.

To define a minimal promoter region, a DNA segment representing thepromoter region is removed from the 5′ region of the gene of interestand operably linked to the coding sequence of a marker (reporter) geneby recombinant DNA techniques well known to the art. The reporter geneis operably linked downstream of the promoter, so that transcriptsinitiating at the promoter proceed through the reporter gene. Reportergenes generally encode proteins which are easily measured, including,but not limited to, chloramphenicol acetyl transferase (CAT),beta-glucuronidase (GUS), green fluorescent protein (GFP),beta-galactosidase (beta-GAL), and luciferase.

The construct containing the reporter gene under the control of thepromoter is then introduced into an appropriate cell type bytransfection techniques well known to the art. To assay for the reporterprotein, cell lysates are prepared and appropriate assays, which arewell known in the art, for the reporter protein are performed. Forexample, if CAT were the reporter gene of choice, the lysates from cellstransfected with constructs containing CAT under the control of apromoter under study are mixed with isotopically labeled chloramphenicoland acetyl-coenzyme A (acetyl-CoA). The CAT enzyme transfers the acetylgroup from acetyl-CoA to the 2- or 3-position of chloramphenicol. Thereaction is monitored by thin-layer chromatography, which separatesacetylated chloramphenicol from unreacted material. The reactionproducts are then visualized by autoradiography.

The level of enzyme activity corresponds to the amount of enzyme thatwas made, which in turn reveals the level of expression from thepromoter of interest. This level of expression can be compared to otherpromoters to determine the relative strength of the promoter understudy. In order to be sure that the level of expression is determined bythe promoter, rather than by the stability of the mRNA, the level of thereporter mRNA can be measured directly, such as by Northern blotanalysis.

Once activity is detected, mutational and/or deletional analyses may beemployed to determine the minimal region and/or sequences required toinitiate transcription. Thus, sequences can be deleted at the 5′ end ofthe promoter region and/or at the 3′ end of the promoter region, andnucleotide substitutions introduced. These constructs are thenintroduced to cells and their activity determined.

In one embodiment, the promoter may be a gamma zein promoter, an oleosinole16 promoter, a globulinI promoter, an actin I promoter, an actin clpromoter, a sucrose synthetase promoter, an INOPS promoter, an EXM5promoter, a globulin2 promoter, a b-32, ADPG-pyrophosphorylase promoter,an LtpI promoter, an Ltp2 promoter, an oleosin ole17 promoter, anoleosin ole18 promoter, an actin 2 promoter, a pollen-specific proteinpromoter, a pollen-specific pectate lyase promoter, an anther-specificprotein promoter, an anther-specific gene RTS2 promoter, apollen-specific gene promoter, a tapeturn-specific gene promoter,tapetum-specific gene RAB24 promoter, a anthranilate synthase alphasubunit promoter, an alpha zein promoter, an anthranilate synthase betasubunit promoter, a dihydrodipicolinate synthase promoter, a Thilpromoter, an alcohol dehydrogenase promoter, a cab binding proteinpromoter, an H3C4 promoter, a RUBISCO SS starch branching enzymepromoter, an ACCase promoter, an actin3 promoter, an actin7 promoter, aregulatory protein GF14-12 promoter, a ribosomal protein L9 promoter, acellulose biosynthetic enzyme promoter, an S-adenosyl-L-homocysteinehydrolase promoter, a superoxide dismutase promoter, a C-kinase receptorpromoter, a phosphoglycerate mutase promoter, a root-specific RCc3 mRNApromoter, a glucose-6 phosphate isomerase promoter, apyrophosphate-fructose 6-phosphatelphosphotransferase promoter, anubiquitin promoter, a beta-ketoacyl-ACP synthase promoter, a 33 kDaphotosystem 11 promoter, an oxygen evolving protein promoter, a 69 kDavacuolar ATPase subunit promoter, a metallothionein-like proteinpromoter, a glyceraldehyde-3-phosphate dehydrogenase promoter, an ABA-and ripening-inducible-like protein promoter, a phenylalanine ammonialyase promoter, an adenosine triphosphatase S-adenosyl-L-homocysteinehydrolase promoter, an α-tubulin promoter, a cab promoter, a PEPCasepromoter, an R gene promoter, a lectin promoter, a light harvestingcomplex promoter, a heat shock protein promoter, a chalcone synthasepromoter, a zein promoter, a globulin-1 promoter, an ABA promoter, anauxin-binding protein promoter, a UDP glucose flavonoidglycosyl-transferase gene promoter, an NTI promoter, an actin promoter,an opaque 2 promoter, a b70 promoter, an oleosin promoter, a CaMV 35Spromoter, a CaMV 19S promoter, a histone promoter, a turgor-induciblepromoter, a pea small subunit RuBP carboxylase promoter, a Ti plasmidmannopine synthase promoter, Ti plasmid nopaline synthase promoter, apetunia chalcone isomerase promoter, a bean glycine rich protein Ipromoter, a CaMV 35S transcript promoter, a potato patatin promoter, ora S-E9 small subunit RuBP carboxylase promoter.

In some embodiments, a promoter has a nucleic acid sequence as set forthin one of SEQ ID NOs: 1-26.

2. Other Regulatory Elements

In addition to promoters, a variety of 5′ and 3′ transcriptionalregulatory sequences are also available for use in the presentinvention. Transcriptional terminators are responsible for thetermination of transcription and correct mRNA polyadenylation. The 3′nontranslated regulatory DNA sequence preferably includes from about 50to about 1,000, more preferably about 100 to about 1,000, nucleotidebase pairs and contains plant transcriptional and translationaltermination sequences. Appropriate transcriptional terminators and thosewhich are known to function in plants include the CaMV 35S terminator,the tml terminator, the nopaline synthase terminator, the pea rbcS E9terminator, the terminator for the T7 transcript from the octopinesynthase gene of Agrobacterium tumefaciens, and the 3′ end of theprotease inhibitor I or II genes from potato or tomato, although other3′ elements known to those of skill in the art can also be employed.Alternatively, one also could use a gamma coixin, oleosin 3 or otherterminator from the genus Coix.

Preferred 3′ elements include those from the nopaline synthase gene ofAgrobacterium tumefaciens (Bevan et al., 1983), the terminator for theT7 transcript from the octopine synthase gene of Agrobacteriumtumefaciens, and the 3′ end of the protease inhibitor I or II genes frompotato or tomato.

As the DNA sequence between the transcription initiation site and thestart of the coding sequence, i.e., the untranslated leader sequence,can influence gene expression, one may also wish to employ a particularleader sequence. Preferred leader sequences are contemplated to includethose which include sequences predicted to direct optimum expression ofthe attached gene, i.e., to include a preferred consensus leadersequence which may increase or maintain mRNA stability and preventinappropriate initiation of translation. The choice of such sequenceswill be known to those of skill in the art in light of the presentdisclosure. Sequences that are derived from genes that are highlyexpressed in plants will be most preferred.

Other sequences that have been found to enhance gene expression intransgenic plants include intron sequences (e.g., from Adh1, bronze1,actin1, actin 2 (WO 00/760067), or the sucrose synthase intron) andviral leader sequences (e.g., from TMV, MCMV and AMV). For example, anumber of non-translated leader sequences derived from viruses are knownto enhance expression. Specifically, leader sequences from TobaccoMosaic Virus (TMV), Maize Chlorotic Mottle Virus (MCMV), and AlfalfaMosaic Virus (AMV) have been shown to be effective in enhancingexpression (e.g., Gallie et al., 1987; Skuzeski et al., 1990). Otherleaders known in the art include but are not limited to: Picornavirusleaders, for example, EMCV leader (Encephalomyocarditis 5 noncodingregion) (Elroy-Stein et al., 1989); Potyvirus leaders, for example, TEVleader (Tobacco Etch Virus); MDMV leader (Maize Dwarf Mosaic Virus);Human immunoglobulin heavy-chain binding protein (BiP) leader, (Macejaket al., 1991); Untranslated leader from the coat protein mRNA of alfalfamosaic virus (AMV RNA 4), (Jobling et al., 1987; Tobacco mosaic virusleader (TMV), (Gallie et al., 1989; and Maize Chlorotic Mottle Virusleader (MCMV) (Lommel et al., 1991. See also, Della-Cioppa et al., 1987.

Regulatory elements such as Adh intron 1 (Callis et al., 1987), sucrosesynthase intron (Vasil et al., 1989) or TMV omega element (Gallie, etal., 1989), may further be included where desired.

Examples of enhancers include elements from the CaMV 35S promoter,octopine synthase genes (Ellis et al., 1987), the rice actin I gene, themaize alcohol dehydrogenase gene (Callis et al., 1987), the maizeshrunken I gene (Vasil et al., 1989), TMV Omega element (Gallie et al.,1989) and promoters from non-plant eukaryotes (e.g. yeast; Ma et al.,1988).

Vectors for use in accordance with the present invention may beconstructed to include the ocs enhancer element. This element was firstidentified as a 16 bp palindromic enhancer from the octopine synthase(ocs) gene of ultilane (Ellis et al., 1987), and is present in at least10 other promoters (Bouchez et al., 1989). The use of an enhancerelement, such as the ocs element and particularly multiple copies of theelement, will act to increase the level of transcription from adjacentpromoters when applied in the context of monocot transformation.

Ultimately, the most desirable DNA segments for introduction into forexample a monocot genome may be homologous genes or gene families whichencode a desired trait (e.g., increased yield per acre) and which areintroduced under the control of novel promoters or enhancers, etc., orperhaps even homologous or tissue specific (e.g., root-, collar/sheath-,whorl-, stalk-, earshank-, kernel- or leaf-specific) promoters orcontrol elements. Indeed, it is envisioned that a particular use of thepresent invention will be the targeting of a gene in a constitutivemanner or a root-specific manner. For example, insect resistant genesmay be expressed specifically in the whorl and collar/sheath tissueswhich are targets for the first and second broods, respectively, of ECB.Likewise, genes encoding proteins with particular activity againstrootworm may be targeted directly to root tissues.

Vectors for use in tissue-specific targeting of genes in transgenicplants will typically include tissue-specific promoters and may alsoinclude other tissue-specific control elements such as enhancersequences. Promoters which direct specific or enhanced expression incertain plant tissues will be known to those of skill in the art inlight of the present disclosure. These include, for example, the rbcSpromoter, specific for green tissue; the ocs, nos and mas promoterswhich have higher activity in roots or wounded leaf tissue; a truncated(−90 to +8) 35S promoter which directs enhanced expression in roots, analpha-tubulin gene that directs expression in roots and promotersderived from zein storage protein genes which direct expression inendosperm. It is particularly contemplated that one may advantageouslyuse the 16 bp ocs enhancer element from the octopine synthase (ocs) gene(Ellis et al., 1987; Bouchez et al., 1989), especially when present inmultiple copies, to achieve enhanced expression in roots.

Tissue specific expression may be functionally accomplished byintroducing a constitutively expressed gene (all tissues) in combinationwith an antisense gene that is expressed only in those tissues where thegene product is not desired. For example, a gene coding for the crystaltoxin protein from B. thuringiensis (Bt) may be introduced such that itis expressed in all tissues using the 35S promoter from CauliflowerMosaic Virus. Expression of an antisense transcript of the Bt gene in amaize kernel, using for example a zein promoter, would preventaccumulation of the Bt protein in seed. Hence the protein encoded by theintroduced gene would be present in all tissues except the kernel.

Expression of some genes in transgenic plants will be desired only underspecified conditions. For example, it is proposed that expression ofcertain genes that confer resistance to environmental stress factorssuch as drought will be desired only under actual stress conditions. Itis contemplated that expression of such genes throughout a plantsdevelopment may have detrimental effects. It is known that a largenumber of genes exist that respond to the environment. For example,expression of some genes such as rbcS, encoding the small subunit ofribulose bisphosphate carboxylase, is regulated by light as mediatedthrough phytochrome. Other genes are induced by secondary stimuli. Forexample, synthesis of abscisic acid (ABA) is induced by certainenvironmental factors, including but not limited to water stress. Anumber of genes have been shown to be induced by ABA (Skriver and Mundy,1990). It is also anticipated that expression of genes conferringresistance to insect predation would be desired only under conditions ofactual insect infestation. Therefore, for some desired traits inducibleexpression of genes in transgenic plants will be desired.

Expression of a gene in a transgenic plant will be desired only in acertain time period during the development of the plant. Developmentaltiming is frequently correlated with tissue specific gene expression.For example, expression of zein storage proteins is initiated in theendosperm about 15 days after pollination.

Additionally, vectors may be constructed and employed in theintracellular targeting of a specific gene product within the cells of atransgenic plant or in directing a protein to the extracellularenvironment. This will generally be achieved by joining a DNA sequenceencoding a transit or signal peptide sequence to the coding sequence ofa particular gene. The resultant transit, or signal, peptide willtransport the protein to a particular intracellular, or extracellulardestination, respectively, and will then be post-translationallyremoved. Transit or signal peptides act by facilitating the transport ofproteins through intracellular membranes, e.g., vacuole, vesicle,plastid and mitochondrial membranes, whereas signal peptides directproteins through the extracellular membrane.

A particular example of such a use concerns the direction of a herbicideresistance gene, such as the EPSPS gene, to a particular organelle suchas the chloroplast rather than to the cytoplasm. This is exemplified bythe use of the rbcs transit peptide which confers plastid-specifictargeting of proteins. In addition, it is proposed that it may bedesirable to target certain genes responsible for male sterility to themitochondria, or to target certain genes for resistance tophytopathogenic organisms to the extracellular spaces, or to targetproteins to the vacuole.

By facilitating the transport of the protein into compartments insideand outside the cell, these sequences may increase the accumulation ofgene product protecting them from proteolytic degradation. Thesesequences also allow for additional mRNA sequences from highly expressedgenes to be attached to the coding sequence of the genes. Since mRNAbeing translated by ribosomes is more stable than naked mRNA, thepresence of translatable mRNA in front of the gene may increase theoverall stability of the mRNA transcript from the gene and therebyincrease synthesis of the gene product. Since transit and signalsequences are usually post-translationally removed from the initialtranslation product, the use of these sequences allows for the additionof extra translated sequences that may not appear on the finalpolypeptide. Targeting of certain proteins may be desirable in order toenhance the stability of the protein (U.S. Pat. No. 5,545,818).

It may be useful to target DNA itself within a cell. For example, it maybe useful to target introduced DNA to the nucleus as this may increasethe frequency of transformation. Within the nucleus itself it would beuseful to target a gene in order to achieve site specific integration.For example, it would be useful to have an gene introduced throughtransformation replace an existing gene in the cell.

Other elements include those that can be regulated by endogenous orexogenous agents, e.g., by zinc finger proteins, including naturallyoccurring zinc finger proteins or chimeric zinc finger proteins (see,e.g., U.S. Pat. No. 5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO98/53057; WO 98/53058; WO 00/23464; WO 95/19431; and WO 98/54311) ormyb-like transcription factors. For example, a chimeric zinc fingerprotein may include amino acid sequences which bind to a specific DNAsequence (the zinc finger) and amino acid sequences that activate (e.g.,GAL 4 sequences) or repress the transcription of the sequences linked tothe specific DNA sequence.

3. Preferred Nucleic Acid Molecules of the Invention

The invention relates to an isolated plant, e.g., Arabidopsis and rice,nucleic acid molecule, which directs the expression of linked nucleicacid fragment in a plant, e.g., in root or leaf or constitutively, aswell as the corresponding open reading frame and encoded product. Thenucleic acid molecule, e.g., one which comprises a promoter can be usedto overexpress a linked nucleic acid fragment so as to express a productin a constitutive or tissue-specific manner, or to alter the expressionof the product, e.g., via the use of antisense vectors or by “knockingout” the expression of at least one genomic copy of the gene.

Preferred sources from which the nucleic acid molecules of the inventioncan be obtained or isolated include, but are not limited to, corn (Zeamays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularlythose Brassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea ultilane),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, duckweed (Lemna), barley, vegetables, ornamentals, and conifers.

Duckweed (Lemna, see WO 00/07210) includes members of the familyLemnaceae. There are known four genera and 34 species of duckweed asfollows: genus Lemna (L. aequinoctialis, L. disperma, L. ecuadoriensis,L. gibba, L. japonica, L. minor, L. miniscula, L. obscura, L.perpusilli, L. tenera, L. trisulca, L. turionifera, L. valdiviana);genus Spirodela (S. intermedia, S. polyrrhiza, S. punctata); genusWoffia (Wa. Angusta, Wa. Arrhiza, Wa. Australina, Wa. Borealis, Wa.Brasiliensis, Wa. Columbiana, Wa. Elongata, Wa. Globosa, Wa.Microscopica, Wa. Neglecta) and genus Wofiella (W1. ultila, W1. ultilanen, W1. gladiata, W1. ultila, W1. lingulata, W1. repunda, W1. rotunda,and W1. neotropica). Any other genera or species of Lemnaceae, if theyexist, are also aspects of the present invention. Lemna gibba, Lemnaminor, and Lemna miniscula are preferred, with Lemna minor and Lemnaminiscula being most preferred. Lemna species can be classified usingthe taxonomic scheme described by Landolt, Biosystematic Investigationon the Family of Duckweeds: The family of Lemnaceae—A Monograph Study.Geobatanischen Institut ETH, Stiftung Rubel, Zurich (1986)).

Vegetables from which to obtain or isolate the nucleic acid molecules ofthe invention include, but are not limited to, tomatoes (Lycopersiconesculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolusvulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), andmembers of the genus Cucumis such as cucumber (C. sativus), cantaloupe(C. cantalupensis), and musk melon (C. melo). Ornamentals from which toobtain or isolate the nucleic acid molecules of the invention include,but are not limited to, azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the present invention include, for example, pinessuch as loblolly pine (Pinus taeda), slash pine (Pinus elliotii),ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), andMonterey pine (Pinus radiata), Douglas-fir (Pseudotsuga menziesii);Western hemlock (Tsuga ultilane); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Leguminous plants from which the nucleic acid molecules of the inventioncan be isolated or obtained include, but are not limited to, beans andpeas. Beans include guar, locust bean, fenugreek, soybean, garden beans,cowpea, mungbean, lima bean, fava bean, lentils, chickpea, and the like.Legumes include, but are not limited to, Arachis, e.g., peanuts, Vicia,e.g., crown vetch, hairy vetch, adzuki bean, mung bean, and chickpea,Lupinus, e.g., lupine, trifolium, Phaseolus, e.g., common bean and limabean, Pisum, e.g., field bean, Melilotus, e.g., clover, Medicago, e.g.,alfalfa, Lotus, e.g., trefoil, lens, e.g., lentil, and false indigo.Preferred forage and turf grass from which the nucleic acid molecules ofthe invention can be isolated or obtained for use in the methods of theinvention include, but are not limited to, alfalfa, orchard grass, tallfescue, perennial ryegrass, creeping bent grass, and redtop.

Other preferred sources of the nucleic acid molecules of the inventioninclude Acacia, aneth, artichoke, arugula, blackberry, canola, cilantro,clementines, escarole, eucalyptus, fennel, grapefruit, honey dew,jicama, kiwifruit, lemon, lime, mushroom, nut, okra, orange, parsley,persimmon, plantain, pomegranate, poplar, radiata pine, radicchio,Southern pine, sweetgum, tangerine, triticale, vine, yams, apple, pear,quince, cherry, apricot, melon, hemp, buckwheat, grape, raspberry,chenopodium, blueberry, nectarine, peach, plum, strawberry, watermelon,eggplant, pepper, cauliflower, Brassica, e.g., broccoli, cabbage,ultilan sprouts, onion, carrot, leek, beet, broad bean, celery, radish,pumpkin, endive, gourd, garlic, snapbean, spinach, squash, turnip,ultilane, and zucchini.

Yet other sources of nucleic acid molecules are ornamental plantsincluding, but not limited to, impatiens, Begonia, Pelargonium, Viola,Cyclamen, Verbena, Vinca, Tagetes, Primula, Saint Paulia, Agertum,Amaranthus, Antihirrhinum, Aquilegia, Cineraria, Clover, Cosmo, Cowpea,Dahlia, Datura, Delphinium, Gerbera, Gladiolus, Gloxinia, Hippeastrum,Mesembryanthemum, Salpiglossos, and Zinnia, and plants such as thoseshown in Table 1.

TABLE 1 MAP REFERENCES FAMILY LATIN NAME COMMON NAME RESOURCESCucurbitaceae Cucumis sativus Cucumber Cucumis melo Melon Citrulluslanatus Watermelon Cucurbita pepo Squash - summer Cucurbita maximaSquash - winter Cucurbita moschata Pumpkin/ butternut SolanaceaeLycopersicon Tomato 15x BAC on variety Heinz esculentum 1706 order fromClemson Genome center 11.6x BAC of L. cheesmanii (originates from J.Giovannoni) available from Clemson genome center EST collection fromTIGR EST collection from Clemsom Genome Center TAG 99: 254-271, 1999(esculentum x pennelli) TAG 89: 1007-1013, 1994 (peruvianum) Plant CellReports 12: 293-297, 1993 (RAPDs) Genetics 132: 1141-1160, 1992 (potatox tomato) Genetics 120: 1095-1105, 1988 (RFLP potato and tomato)Genetics 115: 387-393, 1986 (esculentum x pennelli isozyme and cDNAs)Capsicum annuum Pepper Capsicum frutescens Chile pepper Solanummelongena Eggplant (Nicotiana tabacum) (Tobacco) (Solanum tuberosum)(Potato) (Petunia x (Petunia) 4x BAC of Petunia hybrida hybrida hort.7984 available from Clemson Ex E. Vilm.) genome center BrassicaceaeBrassica oleracea Broccoli L. var. italica Brassica oleracea Cabbage L.var. capitata Brassica rapa Chinese Cabbage Brassica oleraceaCauliflower L. var. botrytis Raphanus sativus Daikon var. niger(Brassica napus) (Oilseed rape) Arabidopsis 12x and 6x BACs on Columbiastrain available from Clemson genome center Umbelliferae Daucus carotaCarrot Compositae Lactuca sativa Lettuce Helianthus annuus (Sunflower)Chenopodiaceae Spinacia oleracea Spinach (Beta vulgaris) (Sugar Beet)Leguminosae Phaseolus vulgaris Bean 4.3x BAC available from Clemsongenome center Pisum sativum Pea (Glycine max) (Soybean) 7.5x and 7.9xBACs available from Clemson genome center Gramineae Zea mays Sweet CornNovartis BACs for Mo17 and B73 have been donated to Clemson GenomeCenter (Zea mays) (Field Corn) Liliaceae Allium cepa Onion Leek (Garlic)(Asparagus)Preferred forage and turf grass nucleic acid sources for the nucleicacid molecules of the invention include, but are not limited to,alfalfa, orchard grass, tall fescue, perennial ryegrass, creeping bentgrass, and redtop. Yet other preferred sources include, but are notlimited to, crop plants and in particular cereals (for example, corn,alfalfa, sunflower, rice, Brassica, canola, soybean, barley, soybean,sugarbeet, cotton, safflower, peanut, sorghum, wheat, millet, tobacco,and the like), and even more preferably corn, rice and soybean.

Based on the Arabidopsis nucleic acid sequence of the present invention,orthologs may be identified or isolated from the genome of any desiredorganism, preferably from another plant, according to well knowntechniques based on their sequence similarity to the Arabidopsis nucleicacid sequences, e.g., hybridization, PCR or computer generated sequencecomparisons. For example, all or a portion of a particular Arabidopsisnucleic acid sequence is used as a probe that selectively hybridizes toother gene sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen source organism. Further, suitable genomic and cDNA libraries maybe prepared from any cell or tissue of an organism. Such techniquesinclude hybridization screening of plated DNA libraries (either plaquesor colonies; see, e.g., Sambrook et al., 1989) and amplification by PCRusing oligonucleotide primers preferably corresponding to sequencedomains conserved among related polypeptide or subsequences of thenucleotide sequences provided herein (see, e.g., Innis et al., 1990).These methods are particularly well suited to the isolation of genesequences from organisms closely related to the organism from which theprobe sequence is derived. The application of these methods using theArabidopsis sequences as probes is well suited for the isolation of genesequences from any source organism, preferably other plant species. In aPCR approach, oligonucleotide primers can be designed for use in PCRreactions to amplify corresponding DNA sequences from cDNA or genomicDNA extracted from any plant of interest. Methods for designing PCRprimers and PCR cloning are generally known in the art.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the sequence of theinvention. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed in Sambrook et al. (1989). In general, sequencesthat hybridize to the sequences disclosed herein will have at least 40%to 50%, about 60% to 70% and even about 80% 85%, 90%, 95% to 98% or moreidentity with the disclosed sequences. That is, the sequence similarityof sequences may range, sharing at least about 40% to 50%, about 60% to70%, and even about 80%, 85%, 90%, 95% to 98% sequence similarity.

The nucleic acid molecules of the invention can also be identified by,for example, a search of known databases for genes encoding polypeptideshaving a specified amino acid sequence identity or DNA having aspecified nucleotide sequence identity. Methods of alignment ofsequences for comparison are well known in the art and are describedherein.

For example, to identify orthologs of the sequences described herein,similarity searches were carried out in databases using a BLAST (seeabove) algorithm followed by analysis using SCAN (the SequenceComparison Analysis, program version 1.0 k licensed from the Los AlmosNational Laboratories) software with added filters.

4. Methods for Mutagenizing Regulatory Elements

It is specifically contemplated by the inventors that one couldmutagenize a promoter to, for example, potentially improve the utilityof the elements for the expression of transgenes in plants. Themutagenesis of these elements can be carried out at random and themutagenized promoter sequences screened for activity in a trial-by-errorprocedure.

Alternatively, particular sequences which provide the promoter withdesirable expression characteristics, or the promoter with expressionenhancement activity, could be identified and these or similar sequencesintroduced into the sequences via mutation. It is further contemplatedthat one could mutagenize these sequences in order to enhance theirexpression of transgenes in a particular species.

The means for mutagenizing a DNA segment encoding a promoter sequence ofthe current invention are well-known to those of skill in the art. Asindicated, modifications to promoter or other regulatory element may bemade by random, or site-specific mutagenesis procedures. The promoterand other regulatory element may be modified by altering their structurethrough the addition or deletion of one or more nucleotides from thesequence which encodes the corresponding unmodified sequences.

Mutagenesis may be performed in accordance with any of the techniquesknown in the art, such as, and not limited to, synthesizing anoligonucleotide having one or more mutations within the sequence of aparticular regulatory region. In particular, site-specific mutagenesisis a technique useful in the preparation of promoter mutants, throughspecific mutagenesis of the underlying DNA. The technique furtherprovides a ready ability to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 toabout 75 nucleotides or more in length is preferred, with about 10 toabout 25 or more residues on both sides of the junction of the sequencebeing altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art.

Double stranded plasmids also are routinely employed in site directedmutagenesis which eliminates the step of transferring the gene ofinterest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the promoter. An oligonucleotideprimer bearing the desired mutated sequence is prepared, generallysynthetically. This primer is then annealed with the single-strandedvector, and subjected to DNA polymerizing enzymes such as E. colipolymerase I Klenow fragment, in order to complete the synthesis of themutation-bearing strand. Thus, a heteroduplex is formed wherein onestrand encodes the original non-mutated sequence and the second strandbears the desired mutation.

This heteroduplex vector is then used to transform or transfectappropriate cells, such as E. coli cells, and cells are selected whichinclude recombinant vectors bearing the mutated sequence arrangement.Vector DNA can then be isolated from these cells and used for planttransformation. A genetic selection scheme was devised by Kunkel et al.(1987) to enrich for clones incorporating mutagenic oligonucleotides.Alternatively, the use of PCR with commercially available thermostableenzymes such as Taq polymerase may be used to incorporate a mutagenicoligonucleotide primer into an amplified DNA fragment that can then becloned into an appropriate cloning or expression vector. ThePCR-mediated mutagenesis procedures of Tomic et al. (1990) and Upenderet al. (1995) provide two examples of such protocols. A PCR employing athermostable ligase in addition to a thermostable polymerase also may beused to incorporate a phosphorylated mutagenic oligonucleotide into anamplified DNA fragment that may then be cloned into an appropriatecloning or expression vector. The mutagenesis procedure described byMichael (1994) provides an example of one such protocol.

The preparation of sequence variants of the selected promoter-encodingDNA segments using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of DNA sequences may beobtained. For example, recombinant vectors encoding the desired promotersequence may be treated with mutagenic agents, such as hydroxylamine, toobtain sequence variants.

In addition, an unmodified or modified nucleotide sequence of thepresent invention can be varied by shuffling the sequence of theinvention. To test for a function of variant DNA sequences according tothe invention, the sequence of interest is operably linked to aselectable or screenable marker gene and expression of the marker geneis tested in transient expression assays with protoplasts or in stablytransformed plants. It is known to the skilled artisan that DNAsequences capable of driving expression of an associated nucleotidesequence are build in a modular way. Accordingly, expression levels fromshorter DNA fragments may be different than the one from the longestfragment and may be different from each other. For example, deletion ofa down-regulating upstream element will lead to an increase in theexpression levels of the associated nucleotide sequence while deletionof an up-regulating element will decrease the expression levels of theassociated nucleotide sequence. It is also known to the skilled artisanthat deletion of development-specific or a tissue-specific element willlead to a temporally or spatially altered expression profile of theassociated nucleotide sequence.

Embraced by the present invention are also functional equivalents of thepromoters of the present invention, i.e. nucleotide sequences thathybridize under stringent conditions to the reverse-complement of anyone of SEQ ID NOs: 1-26, or the promoter orthologs thereof.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” also is intended to refer to a process thatinvolves the template-dependent extension of a primer molecule. The termtemplate-dependent process refers to nucleic acid synthesis of an RNA ora DNA molecule wherein the sequence of the newly synthesized strand ofnucleic acid is dictated by the well-known rules of complementary basepairing (see, for example, Watson and Rarnstad, 1987). Typically, vectormediated methodologies involve the introduction of the nucleic acidfragment into a DNA or RNA vector, the clonal amplification of thevector, and the recovery of the amplified nucleic acid fragment.Examples of such methodologies are provided by U.S. Pat. No. 4,237,224.A number of template dependent processes are available to amplify thetarget sequences of interest present in a sample, such methods beingwell known in the art and specifically disclosed herein below.

Where a clone comprising a promoter has been isolated in accordance withthe instant invention, one may wish to delimit the essential promoterregions within the clone. One efficient, targeted means for preparingmutagenizing promoters relies upon the identification of putativeregulatory elements within the promoter sequence. This can be initiatedby comparison with promoter sequences known to be expressed in similartissue-specific or developmentally unique manner. Sequences which areshared among promoters with similar expression patterns are likelycandidates for the binding of transcription factors and are thus likelyelements which confer expression patterns. Confirmation of theseputative regulatory elements can be achieved by deletion analysis ofeach putative regulatory region followed by functional analysis of eachdeletion construct by assay of a reporter gene which is functionallyattached to each construct. As such, once a starting promoter sequenceis provided, any of a number of different deletion mutants of thestarting promoter could be readily prepared.

As indicated above, deletion mutants, deletion mutants of the promoterof the invention also could be randomly prepared and then assayed. Withthis strategy, a series of constructs are prepared, each containing adifferent portion of the clone (a subclone), and these constructs arethen screened for activity. A suitable means for screening for activityis to attach a deleted promoter or intron construct which contains adeleted segment to a selectable or screenable marker, and to isolateonly those cells expressing the marker gene. In this way, a number ofdifferent, deleted promoter constructs are identified which still retainthe desired, or even enhanced, activity. The smallest segment which isrequired for activity is thereby identified through comparison of theselected constructs. This segment may then be used for the constructionof vectors for the expression of exogenous genes.

B. Marker Genes

In order to improve the ability to identify transformants, one maydesire to employ a selectable or screenable marker gene as, or inaddition to, the expressible gene of interest. “Marker genes” are genesthat impart a distinct phenotype to cells expressing the marker gene andthus allow such transformed cells to be distinguished from cells that donot have the marker. Such genes may encode either a selectable orscreenable marker, depending on whether the marker confers a trait whichone can ‘select’ for by chemical means, i.e., through the use of aselective agent (e.g., a herbicide, antibiotic, or the like), or whetherit is simply a trait that one can identify through observation ortesting, i.e., by ‘screening’ (e.g., the R-locus trait, the greenfluorescent protein (GFP)). Of course, many examples of suitable markergenes are known to the art and can be employed in the practice of theinvention.

Included within the terms selectable or screenable marker genes are alsogenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which encode a secretable antigen that can be identifiedby antibody interaction, or even secretable enzymes which can bedetected by their catalytic activity. Secretable proteins fall into anumber of classes, including small, diffusible proteins detectable,e.g., by ELISA; small active enzymes detectable in extracellularsolution (e.g., alpha-amylase, beta-lactamase, phosphinothricinacetyltransferase); and proteins that are inserted or trapped in thecell wall (e.g., proteins that include a leader sequence such as thatfound in the expression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene thatencodes a protein that becomes sequestered in the cell wall, and whichprotein includes a unique epitope is considered to be particularlyadvantageous. Such a secreted antigen marker would ideally employ anepitope sequence that would provide low background in plant tissue, apromoter-leader sequence that would impart efficient expression andtargeting across the plasma membrane, and would produce protein that isbound in the cell wall and yet accessible to antibodies. A normallysecreted wall protein modified to include a unique epitope would satisfyall such requirements.

One example of a protein suitable for modification in this manner isextensin, or hydroxyproline rich glycoprotein (HPRG). For example, themaize HPRG (Steifel et al., 1990) molecule is well characterized interms of molecular biology, expression and protein structure. However,any one of a variety of ultilane and/or glycine-rich wall proteins(Keller et al., 1989) could be modified by the addition of an antigenicsite to create a screenable marker.

One exemplary embodiment of a secretable screenable marker concerns theuse of a maize sequence encoding the wall protein HPRG, modified toinclude a 15 residue epitope from the pro-region of murine interleukin,however, virtually any detectable epitope may be employed in suchembodiments, as selected from the extremely wide variety ofantigen-antibody combinations known to those of skill in the art. Theunique extracellular epitope can then be straightforwardly detectedusing antibody labeling in conjunction with chromogenic or fluorescentadjuncts.

Elements of the present disclosure may be exemplified in detail throughthe use of the bar and/or GUS genes, and also through the use of variousother markers. Of course, in light of this disclosure, numerous otherpossible selectable and/or screenable marker genes will be apparent tothose of skill in the art in addition to the one set forth hereinbelow.Therefore, it will be understood that the following discussion isexemplary rather than exhaustive. In light of the techniques disclosedherein and the general recombinant techniques which are known in theart, the present invention renders possible the introduction of anygene, including marker genes, into a recipient cell to generate atransformed plant.

1. Selectable Markers

Possible selectable markers for use in connection with the presentinvention include, but are not limited to, a neo gene (Potrykus et al.,1985) which codes for kanamycin resistance and can be selected for usingkanamycin, G418, paromomycin, and the like; a bar gene which codes forbialaphos or phosphinothricin resistance; a gene which encodes analtered EPSP synthase protein (Hinchee et al., 1988) thus conferringglyphosate resistance; a nitrilase gene such as bxn from Klebsiellaozaenae which confers resistance to bromoxynil (Stalker et al., 1988); amutant acetolactate synthase gene (ALS) which confers resistance toimidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EuropeanPatent Application 154,204, 1985); a methotrexate-resistant DHFR gene(Thillet et al., 1988); a dalapon dehalogenase gene that confersresistance to the herbicide dalapon; a mutated anthranilate synthasegene that confers resistance to 5-methyl tryptophan. Preferredselectable marker genes encode phosphinothricin acetyltransferase;glyphosate resistant EPSPS, aminoglycoside phosphotransferase;hygromycin phosphotransferase, or neomycin phosphotransferase. Where amutant EPSP synthase gene is employed, additional benefit may berealized through the incorporation of a suitable chloroplast transitpeptide, CTP (European Patent Application 0,218,571, 1987).

An illustrative embodiment of a selectable marker gene capable of beingused in systems to select transformants is the genes that encode theenzyme phosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicus or the pat gene from Streptomycesviridochromogenes. The enzyme phosphinothricin acetyl transferase (PAT)inactivates the active ingredient in the herbicide bialaphos,phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami etal., 1986; Twell et al., 1989) causing rapid accumulation of ammonia andcell death. The success in using this selective system in conjunctionwith monocots was particularly surprising because of the majordifficulties which have been reported in transformation of cereals(Potrykus, 1989).

Where one desires to employ a bialaphos resistance gene in the practiceof the invention, a particularly useful gene for this purpose is the baror pat genes obtainable from species of Streptomyces (e.g., ATCC No.21,705). The cloning of the bar gene has been described (Murakami etal., 1986; Thompson et al., 1987) as has the use of the bar gene in thecontext of plants other than monocots (De Block et al., 1987; De Blocket al., 1989).

Selection markers resulting in positive selection, such as aphosphomannose isomerase gene, as described in patent application WO93/05163, may also be used. Alternative genes to be used for positiveselection are described in WO 94/20627 and encode xyloisomerases andphosphomanno-isomerases such as mannose-6-phosphate isomerase andmannose-1-phosphate isomerase; phosphomanno mutase; mannose epimerasessuch as those which convert carbohydrates to mannose or mannose tocarbohydrates such as glucose or galactose; phosphatases such as mannoseor xylose phosphatase, mannose-6-phosphatase and mannose-1-phosphatase,and permeases which are involved in the transport of mannose, or aderivative, or a precursor thereof into the cell. Transformed cells areidentified without damaging or killing the non-transformed cells in thepopulation and without co-introduction of antibiotic or herbicideresistance genes. As described in WO 93/05163, in addition to the factthat the need for antibiotic or herbicide resistance genes iseliminated, it has been shown that the positive selection method isoften far more efficient than traditional negative selection.

2. Screenable Markers

Screenable markers that may be employed include, but are not limited to,a beta-glucuronidase (GUS) or uidA gene which encodes an enzyme forwhich various chromogenic substrates are known; an R-locus gene, whichencodes a product that regulates the production of anthocyanin pigments(red color) in plant tissues (Dellaporta et al., 1988); a beta-lactamasegene (Sutcliffe, 1978), which encodes an enzyme for which variouschromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a xylE gene (Zukowsky et al., 1983) which encodes acatechol dioxygenase that can convert chromogenic catechols; anα-amylase gene (Ikuta et al., 1990); a tyrosinase gene (Katz et al.,1983) which encodes an enzyme capable of oxidizing tyrosine to DOPA anddopaquinone which in turn condenses to form the easily detectablecompound melanin; a β-galactosidase gene, which encodes an enzyme forwhich there are chromogenic substrates; a luciferase (lux) gene (Ow etal., 1986), which allows for bioluminescence detection; or even anaequorin gene (Prasher et al., 1985), which may be employed incalcium-sensitive bioluminescence detection, or a green fluorescentprotein gene (Niedz et al., 1995).

Genes from the maize R gene complex are contemplated to be particularlyuseful as screenable markers. The R gene complex in maize encodes aprotein that acts to regulate the production of anthocyanin pigments inmost seed and plant tissue. A gene from the R gene complex was appliedto maize transformation, because the expression of this gene intransformed cells does not harm the cells. Thus, an R gene introducedinto such cells will cause the expression of a red pigment and, ifstably incorporated, can be visually scored as a red sector. If a maizeline carries dominant alleles for genes encoding the enzymaticintermediates in the anthocyanin biosynthetic pathway (C2, A1, A2, Bz1and Bz2), but carries a recessive allele at the R locus, transformationof any cell from that line with R will result in red pigment formation.Exemplary lines include Wisconsin 22 which contains the rg-Stadlerallele and TR112, a K55 derivative which is r-g, b, P1. Alternativelyany genotype of maize can be utilized if the C1 and R alleles areintroduced together.

It is further proposed that R gene regulatory regions may be employed inchimeric constructs in order to provide mechanisms for controlling theexpression of chimeric genes. More diversity of phenotypic expression isknown at the R locus than at any other locus (Coe et al., 1988). It iscontemplated that regulatory regions obtained from regions 5′ to thestructural R gene would be valuable in directing the expression ofgenes, e.g., insect resistance, drought resistance, herbicide toleranceor other protein coding regions. For the purposes of the presentinvention, it is believed that any of the various R gene family membersmay be successfully employed (e.g., P, S, Lc, etc.). However, the mostpreferred will generally be Sn (particularly Sn:bol3). Sn is a dominantmember of the R gene complex and is functionally similar to the R and Bloci in that Sn controls the tissue specific deposition of anthocyaninpigments in certain seedling and plant cells, therefore, its phenotypeis similar to R.

A further screenable marker contemplated for use in the presentinvention is firefly luciferase, encoded by the lux gene. The presenceof the lux gene in transformed cells may be detected using, for example,X-ray film, scintillation counting, fluorescent spectrophotometry,low-light video cameras, photon counting cameras or multiwellluminometry. It is also envisioned that this system may be developed forpopulational screening for bioluminescence, such as on tissue cultureplates, or even for whole plant screening. Where use of a screenablemarker gene such as lux or GFP is desired, benefit may be realized bycreating a gene fusion between the screenable marker gene and aselectable marker gene, for example, a GFP-NPTII gene fusion. This couldallow, for example, selection of transformed cells followed by screeningof transgenic plants or seeds.

C. Exogenous Genes for Modification of Plant Phenotypes

Genes of interest are reflective of the commercial markets and interestsof those involved in the development of the crop. Crops and markets ofinterest changes, and as developing nations open up world markets, newcrops and technologies will also emerge. In addition, as theunderstanding of agronomic traits and characteristics such as yield andheterosis increase, the choice of genes for transformation will changeaccordingly. General categories of genes of interest include, forexample, those genes involved in information, such as zinc fingers,those involved in communication, such as kinases, and those involved inhousekeeping, such as heat shock proteins. More specific categories oftransgenes, for example, include genes encoding important traits foragronomics, insect resistance, disease resistance, herbicide resistance,sterility, grain characteristics, and commercial products. Genes ofinterest include, generally, those involved in starch, oil,carbohydrate, or nutrient metabolism, as well as those affecting kernelsize, sucrose loading, zinc finger proteins, see, e.g., U.S. Pat. No.5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO 98/53057; WO98/53058; WO 00/23464; WO 95/19431; and WO 98/54311, and the like.

One skilled in the art recognizes that the expression level andregulation of a transgene in a plant can vary significantly from line toline. Thus, one has to test several lines to find one with the desiredexpression level and regulation. Once a line is identified with thedesired regulation specificity of a chimeric Cre transgene, it can becrossed with lines carrying different inactive replicons or inactivetransgene for activation.

Other sequences which may be linked to the gene of interest whichencodes a polypeptide are those which can target to a specificorganelle, e.g., to the mitochondria, nucleus, or plastid, within theplant cell. Targeting can be achieved by providing the polypeptide withan appropriate targeting peptide sequence, such as a secretory signalpeptide (for secretion or cell wall or membrane targeting, a plastidtransit peptide, a chloroplast transit peptide, e.g., the chlorophylla/b binding protein, a mitochondrial target peptide, a vacuole targetingpeptide, or a nuclear targeting peptide, and the like. For example, thesmall subunit of ribulose bisphosphate carboxylase transit peptide, theEPSPS transit peptide or the dihydrodipicolinic acid synthase transitpeptide may be used. For examples of plastid organelle targetingsequences (see WO 00/12732). Plastids are a class of plant organellesderived from proplastids and include chloroplasts, leucoplasts,aravloplasts, and chromoplasts. The plastids are major sites ofbiosynthesis in plants. In addition to photosynthesis in thechloroplast, plastids are also sites of lipid biosynthesis, nitratereduction to ammonium, and starch storage. And while plastids containtheir own circular genome, most of the proteins localized to theplastids are encoded by the nuclear genome and are imported into theorganelle from the cytoplasm.

Transgenes used with the present invention will often be genes thatdirect the expression of a particular protein or polypeptide product,but they may also be non-expressible DNA segments, e.g., transposonssuch as Ds that do no direct their own transposition. As used herein, an“expressible gene” is any gene that is capable of being transcribed intoRNA (e.g., mRNA, antisense RNA, etc.) or translated into a protein,expressed as a trait of interest, or the like, etc., and is not limitedto selectable, screenable or non-selectable marker genes. The inventionalso contemplates that, where both an expressible gene that is notnecessarily a marker gene is employed in combination with a marker gene,one may employ the separate genes on either the same or different DNAsegments for transformation. In the latter case, the different vectorsare delivered concurrently to recipient cells to maximizecotransformation.

The choice of the particular DNA segments to be delivered to therecipient cells will often depend on the purpose of the transformation.One of the major purposes of transformation of crop plants is to addsome commercially desirable, agronomically important traits to theplant. Such traits include, but are not limited to, herbicide resistanceor tolerance; insect resistance or tolerance; disease resistance ortolerance (viral, bacterial, fungal, nematode); stress tolerance and/orresistance, as exemplified by resistance or tolerance to drought, heat,chilling, freezing, excessive moisture, salt stress; oxidative stress;increased yields; food content and makeup; physical appearance; malesterility; drydown; standability; prolificacy; starch properties; oilquantity and quality; and the like. One may desire to incorporate one ormore genes conferring any such desirable trait or traits, such as, forexample, a gene or genes encoding pathogen resistance.

In certain embodiments, the present invention contemplates thetransformation of a recipient cell with more than one advantageoustransgene. Two or more transgenes can be supplied in a singletransformation event using either distinct transgene-encoding vectors,or using a single vector incorporating two or more gene codingsequences. For example, plasmids bearing the bar and aroA expressionunits in either convergent, divergent, or colinear orientation, areconsidered to be particularly useful. Further preferred combinations arethose of an insect resistance gene, such as a Bt gene, along with aprotease inhibitor gene such as pinII, or the use of bar in combinationwith either of the above genes. Of course, any two or more transgenes ofany description, such as those conferring herbicide, insect, disease(viral, bacterial, fungal, nematode) or drought resistance, malesterility, drydown, standability, prolificacy, starch properties, oilquantity and quality, or those increasing yield or nutritional qualitymay be employed as desired.

1. Herbicide Resistance

The genes encoding phosphinothricin acetyltransferase (bar and pat),glyphosate tolerant EPSP synthase genes, the glyphosate degradativeenzyme gene gox encoding glyphosate oxidoreductase, deh (encoding adehalogenase enzyme that inactivates dalapon), herbicide resistant(e.g., sulfonylurea and imidazolinone) acetolactate synthase, and bxngenes (encoding a nitrilase enzyme that degrades bromoxynil) are goodexamples of herbicide resistant genes for use in transformation. The barand pat genes code for an enzyme, phosphinothricin acetyltransferase(PAT), which inactivates the herbicide phosphinothricin and preventsthis compound from inhibiting glutamine synthetase enzymes. The enzyme5-enolpyruvylshikimate 3-phosphate synthase (EPSP Synthase), is normallyinhibited by the herbicide N-(phosphonomethyl)glycine (glyphosate).However, genes are known that encode glyphosate-resistant EPSP Synthaseenzymes.

These genes are particularly contemplated for use in monocottransformation. The deh gene encodes the enzyme dalapon dehalogenase andconfers resistance to the herbicide dalapon. The bxn gene codes for aspecific nitrilase enzyme that converts bromoxynil to a non-herbicidaldegradation product.

2. Insect Resistance

An important aspect of the present invention concerns the introductionof insect resistance-conferring genes into plants. Potential insectresistance genes which can be introduced include Bacillus thuringiensiscrystal toxin genes or Bt genes (Watrud et al., 1985). Bt genes mayprovide resistance to lepidopteran or coleopteran pests such as EuropeanCorn Borer (ECB) and corn rootworm (CRW). Preferred Bt toxin genes foruse in such embodiments include the CryIA(b) and CryIA(c) genes.Endotoxin genes from other species of B. thuringiensis which affectinsect growth or development may also be employed in this regard.

The poor expression of Bt toxin genes in plants is a well-documentedphenomenon, and the use of different promoters, fusion proteins, andleader sequences has not led to significant increases in Bt proteinexpression (Vaeck et al., 1989; Barton et al., 1987). It is thereforecontemplated that the most advantageous Bt genes for use in thetransformation protocols disclosed herein will be those in which thecoding sequence has been modified to effect increased expression inplants, and more particularly, those in which maize preferred codonshave been used. Examples of such modified Bt toxin genes include thevariant Bt CryIA(b) gene termed lab6 (Perlak et al., 1991) and thesynthetic CryIA(c) genes termed 1800a and 1800b.

Protease inhibitors may also provide insect resistance (Johnson et al.,1989), and will thus have utility in plant transformation. The use of aprotease inhibitor II gene, pinII, from tomato or potato is envisionedto be particularly useful. Even more advantageous is the use of a pinIIgene in combination with a Bt toxin gene, the combined effect of whichhas been discovered by the present inventors to produce synergisticinsecticidal activity. Other genes which encode inhibitors of theinsects' digestive system, or those that encode enzymes or co-factorsthat facilitate the production of inhibitors, may also be useful. Thisgroup may be exemplified by oryzacystatin and amylase inhibitors, suchas those from wheat and barley.

Also, genes encoding lectins may confer additional or alternativeinsecticide properties. Lectins (originally termed phytohemagglutinins)are multivalent carbohydrate-binding proteins which have the ability toagglutinate red blood cells from a range of species. Lectins have beenidentified recently as insecticidal agents with activity againstweevils, ECB and rootworm (Murdock et al., 1990; Czapla and Lang, 1990).Lectin genes contemplated to be useful include, for example, barley andwheat germ agglutinin (WGA) and rice lectins (Gatehouse et al., 1984),with WGA being preferred.

Genes controlling the production of large or small polypeptides activeagainst insects when introduced into the insect pests, such as, e.g.,lytic peptides, peptide hormones and toxins and venoms, form anotheraspect of the invention. For example, it is contemplated that theexpression of juvenile hormone esterase, directed towards specificinsect pests, may also result in insecticidal activity, or perhaps causecessation of metamorphosis (Hammock et al., 1990).

Transgenic plants expressing genes which encode enzymes that affect theintegrity of the insect cuticle form yet another aspect of theinvention. Such genes include those encoding, e.g., chitinase,proteases, lipases and also genes for the production of nikkomycin, acompound that inhibits chitin synthesis, the introduction of any ofwhich is contemplated to produce insect resistant maize plants. Genesthat code for activities that affect insect molting, such thoseaffecting the production of ecdysteroid UDP-glucosyl transferase, alsofall within the scope of the useful transgenes of the present invention.

Genes that code for enzymes that facilitate the production of compoundsthat reduce the nutritional quality of the host plant to insect pestsare also encompassed by the present invention. It may be possible, forinstance, to confer insecticidal activity on a plant by altering itssterol composition. Sterols are obtained by insects from their diet andare used for hormone synthesis and membrane stability. Thereforealterations in plant sterol composition by expression of novel genes,e.g., those that directly promote the production of undesirable sterolsor those that convert desirable sterols into undesirable forms, couldhave a negative effect on insect growth and/or development and henceendow the plant with insecticidal activity. Lipoxygenases are naturallyoccurring plant enzymes that have been shown to exhibit anti-nutritionaleffects on insects and to reduce the nutritional quality of their diet.Therefore, further embodiments of the invention concern transgenicplants with enhanced lipoxygenase activity which may be resistant toinsect feeding.

The present invention also provides methods and compositions by which toachieve qualitative or quantitative changes in plant secondarymetabolites. One example concerns transforming plants to produce DIMBOAwhich, it is contemplated, will confer resistance to European cornborer, rootworm and several other maize insect pests. Candidate genesthat are particularly considered for use in this regard include thosegenes at the bx locus known to be involved in the synthetic DIMBOApathway (Dunn et al., 1981). The introduction of genes that can regulatethe production of maysin, and genes involved in the production ofdhurrin in sorghum, is also contemplated to be of use in facilitatingresistance to earworm and rootworm, respectively.

Tripsacum dactyloides is a species of grass that is resistant to certaininsects, including corn root worm. It is anticipated that genes encodingproteins that are toxic to insects or are involved in the biosynthesisof compounds toxic to insects will be isolated from Tripsacum and thatthese novel genes will be useful in conferring resistance to insects. Itis known that the basis of insect resistance in Tripsacum is genetic,because said resistance has been transferred to Zea mays via sexualcrosses (Branson and Guss, 1972).

Further genes encoding proteins characterized as having potentialinsecticidal activity may also be used as transgenes in accordanceherewith. Such genes include, for example, the cowpea trypsin inhibitor(CpTI; Hilder et al., 1987) which may be used as a rootworm deterrent;genes encoding avermectin (Campbell, 1989; Ikeda et al., 1987) which mayprove particularly useful as a corn rootworm deterrent; ribosomeinactivating protein genes; and even genes that regulate plantstructures. Transgenic maize including anti-insect antibody genes andgenes that code for enzymes that can covert a non-toxic insecticide(pro-insecticide) applied to the outside of the plant into aninsecticide inside the plant are also contemplated.

3. Environment or Stress Resistance

Improvement of a plant's ability to tolerate various environmentalstresses such as, but not limited to, drought, excess moisture,chilling, freezing, high temperature, salt, and oxidative stress, canalso be effected through expression of heterologous, or overexpressionof homologous genes. Benefits may be realized in terms of increasedresistance to freezing temperatures through the introduction of an“antifreeze” protein such as that of the Winter Flounder (Cutler et al.,1989) or synthetic gene derivatives thereof. Improved chilling tolerancemay also be conferred through increased expression ofglycerol-3-phosphate acetyltransferase in chloroplasts (Murata et al.,1992; Wolter et al., 1992). Resistance to oxidative stress (oftenexacerbated by conditions such as chilling temperatures in combinationwith high light intensities) can be conferred by expression ofsuperoxide dismutase (Gupta et al., 1993), and may be improved byglutathione reductase (Bowler et al., 1992). Such strategies may allowfor tolerance to freezing in newly emerged fields as well as extendinglater maturity higher yielding varieties to earlier relative maturityzones.

Expression of novel genes that favorably effect plant water content,total water potential, osmotic potential, and turgor can enhance theability of the plant to tolerate drought. As used herein, the terms“drought resistance” and “drought tolerance” are used to refer to aplants increased resistance or tolerance to stress induced by areduction in water availability, as compared to normal circumstances,and the ability of the plant to function and survive in lower-waterenvironments, and perform in a relatively superior manner. In thisaspect of the invention it is proposed, for example, that the expressionof a gene encoding the biosynthesis of osmotically-active solutes canimpart protection against drought. Within this class of genes are DNAsencoding mannitol dehydrogenase (Lee and Saier, 1982) andtrehalose-6-phosphate synthase (Kaasen et al., 1992). Through thesubsequent action of native phosphatases in the cell or by theintroduction and coexpression of a specific phosphatase, theseintroduced genes will result in the accumulation of either mannitol ortrehalose, respectively, both of which have been well documented asprotective compounds able to mitigate the effects of stress. Mannitolaccumulation in transgenic tobacco has been verified and preliminaryresults indicate that plants expressing high levels of this metaboliteare able to tolerate an applied osmotic stress (Tarczynski et al., citedsupra (1992), 1993).

Similarly, the efficacy of other metabolites in protecting either enzymefunction (e.g. alanopine or propionic acid) or membrane integrity (e.g.,alanopine) has been documented (Loomis et al., 1989), and thereforeexpression of gene encoding the biosynthesis of these compounds canconfer drought resistance in a manner similar to or complimentary tomannitol. Other examples of naturally occurring metabolites that areosmotically active and/or provide some direct protective effect duringdrought and/or desiccation include sugars and sugar derivatives such asfructose, erythritol (Coxson et al., 1992), sorbitol, dulcitol (Karstenet al., 1992), glucosylglycerol (Reed et al., 1984; Erdmann et al.,1992), sucrose, stachyose (Koster and Leopold, 1988; Blackman et al.,1992), ononitol and pinitol (Vernon and Bohnert, 1992), and raffinose(Bernal-Lugo and Leopold, 1992). Other osmotically active solutes whichare not sugars include, but are not limited to, proline andglycine-betaine (Wyn-Jones and Storey, 1981). Continued canopy growthand increased reproductive fitness during times of stress can beaugmented by introduction and expression of genes such as thosecontrolling the osmotically active compounds discussed above and othersuch compounds, as represented in one exemplary embodiment by the enzymemyoinositol O-methyltransferase.

It is contemplated that the expression of specific proteins may alsoincrease drought tolerance. Three classes of Late Embryogenic Proteinshave been assigned based on structural similarities (see Dure et al.,1989). All three classes of these proteins have been demonstrated inmaturing (i.e., desiccating) seeds. Within these 3 types of proteins,the Type-II (dehydrin-type) have generally been implicated in droughtand/or desiccation tolerance in vegetative plant parts (i.e. Mundy andChua, 1988; Piatkowski et al., 1990; Yamaguchi-Shinozaki et al., 1992).Recently, expression of a Type-III LEA (HVA-1) in tobacco was found toinfluence plant height, maturity and drought tolerance (Fitzpatrick,1993). Expression of structural genes from all three groups maytherefore confer drought tolerance. Other types of proteins inducedduring water stress include thiol proteases, aldolases and transmembranetransporters (Guerrero et al., 1990), which may confer variousprotective and/or repair-type functions during drought stress. Theexpression of a gene that effects lipid biosynthesis and hence membranecomposition can also be useful in conferring drought resistance on theplant.

Many genes that improve drought resistance have complementary modes ofaction. Thus, combinations of these genes might have additive and/orsynergistic effects in improving drought resistance in plants. Many ofthese genes also improve freezing tolerance (or resistance); thephysical stresses incurred during freezing and drought are similar innature and may be mitigated in similar fashion. Benefit may be conferredvia constitutive expression of these genes, but the preferred means ofexpressing these novel genes may be through the use of a turgor-inducedpromoter (such as the promoters for the turgor-induced genes describedin Guerrero et al. 1990 and Shagan et al., 1993). Spatial and temporalexpression patterns of these genes may enable maize to better withstandstress.

Expression of genes that are involved with specific morphological traitsthat allow for increased water extractions from drying soil would be ofbenefit. For example, introduction and expression of genes that alterroot characteristics may enhance water uptake. Expression of genes thatenhance reproductive fitness during times of stress would be ofsignificant value. For example, expression of DNAs that improve thesynchrony of pollen shed and receptiveness of the female flower parts,i.e., silks, would be of benefit. In addition, expression of genes thatminimize kernel abortion during times of stress would increase theamount of grain to be harvested and hence be of value. Regulation ofcytokinin levels in monocots, such as maize, by introduction andexpression of an isopentenyl transferase gene with appropriateregulatory sequences can improve monocot stress resistance and yield(Gan et al., Science, 270:1986 (1995)).

Given the overall role of water in determining yield, it is contemplatedthat enabling plants to utilize water more efficiently, through theintroduction and expression of novel genes, will improve overallperformance even when soil water availability is not limiting. Byintroducing genes that improve the ability of plants to maximize waterusage across a full range of stresses relating to water availability,yield stability or consistency of yield performance may be realized.

4. Disease Resistance

It is proposed that increased resistance to diseases may be realizedthrough introduction of genes into plants period. It is possible toproduce resistance to diseases caused by viruses, bacteria, fungi, rootpathogens, insects and nematodes. It is also contemplated that controlof mycotoxin producing organisms may be realized through expression ofintroduced genes.

Resistance to viruses may be produced through expression of novel genes.For example, it has been demonstrated that expression of a viral coatprotein in a transgenic plant can impart resistance to infection of theplant by that virus and perhaps other closely related viruses (Cuozzo etal., 1988, Hemenway et al., 1988, Abel et al., 1986). It is contemplatedthat expression of antisense genes targeted at essential viral functionsmay impart resistance to said virus. For example, an antisense genetargeted at the gene responsible for replication of viral nucleic acidmay inhibit said replication and lead to resistance to the virus. It isbelieved that interference with other viral functions through the use ofantisense genes may also increase resistance to viruses. Further it isproposed that it may be possible to achieve resistance to virusesthrough other approaches, including, but not limited to the use ofsatellite viruses.

It is proposed that increased resistance to diseases caused by bacteriaand fungi may be realized through introduction of novel genes. It iscontemplated that genes encoding so-called “peptide antibiotics,”pathogenesis related (PR) proteins, toxin resistance, and proteinsaffecting host-pathogen interactions such as morphologicalcharacteristics will be useful. Peptide antibiotics are polypeptidesequences which are inhibitory to growth of bacteria and othermicroorganisms. For example, the classes of peptides referred to ascecropins and magainins inhibit growth of many species of bacteria andfungi. It is proposed that expression of PR proteins in plants may beuseful in conferring resistance to bacterial disease. These genes areinduced following pathogen attack on a host plant and have been dividedinto at least five classes of proteins (Bol et al., 1990). Includedamongst the PR proteins are beta-1,3-glucanases, chitinases, and osmotinand other proteins that are believed to function in plant resistance todisease organisms. Other genes have been identified that have antifungalproperties, e.g., UDA (stinging nettle lectin) and hevein (Broakgert etal., 1989; Barkai-Golan et al., 1978). It is known that certain plantdiseases are caused by the production of phytotoxins. Resistance tothese diseases could be achieved through expression of a novel gene thatencodes an enzyme capable of degrading or otherwise inactivating thephytotoxin. Expression novel genes that alter the interactions betweenthe host plant and pathogen may be useful in reducing the ability thedisease organism to invade the tissues of the host plant, e.g., anincrease in the waxiness of the leaf cuticle or other morphologicalcharacteristics.

Plant parasitic nematodes are a cause of disease in many plants. It isproposed that it would be possible to make the plant resistant to theseorganisms through the expression of novel genes. It is anticipated thatcontrol of nematode infestations would be accomplished by altering theability of the nematode to recognize or attach to a host plant and/orenabling the plant to produce nematicidal compounds, including but notlimited to proteins.

5. Mycotoxin Reduction/Elimination

Production of mycotoxins, including aflatoxin and fumonisin, by fungiassociated with plants is a significant factor in rendering the grainnot useful. These fungal organisms do not cause disease symptoms and/orinterfere with the growth of the plant, but they produce chemicals(mycotoxins) that are toxic to animals. Inhibition of the growth ofthese fungi would reduce the synthesis of these toxic substances and,therefore, reduce grain losses due to mycotoxin contamination. Novelgenes may be introduced into plants that would inhibit synthesis of themycotoxin without interfering with fungal growth. Expression of a novelgene which encodes an enzyme capable of rendering the mycotoxin nontoxicwould be useful in order to achieve reduced mycotoxin contamination ofgrain. The result of any of the above mechanisms would be a reducedpresence of mycotoxins on grain.

6. Grain Composition or Quality

Genes may be introduced into plants, particularly commercially importantcereals such as maize, wheat or rice, to improve the grain for which thecereal is primarily grown. A wide range of novel transgenic plantsproduced in this manner may be envisioned depending on the particularend use of the grain.

For example, the largest use of maize grain is for feed or food.Introduction of genes that alter the composition of the grain maygreatly enhance the feed or food value. The primary components of maizegrain are starch, protein, and oil. Each of these primary components ofmaize grain may be improved by altering its level or composition.Several examples may be mentioned for illustrative purposes but in noway provide an exhaustive list of possibilities.

The protein of many cereal grains is suboptimal for feed and foodpurposes especially when fed to pigs, poultry, and humans. The proteinis deficient in several amino acids that are essential in the diet ofthese species, requiring the addition of supplements to the grain.Limiting essential amino acids may include lysine, methionine,tryptophan, threonine, valine, arginine, and histidine. Some amino acidsbecome limiting only after the grain is supplemented with other inputsfor feed formulations. For example, when the grain is supplemented withsoybean meal to meet lysine requirements, methionine becomes limiting.The levels of these essential amino acids in seeds and grain may beelevated by mechanisms which include, but are not limited to, theintroduction of genes to increase the biosynthesis of the amino acids,decrease the degradation of the amino acids, increase the storage of theamino acids in proteins, or increase transport of the amino acids to theseeds or grain.

One mechanism for increasing the biosynthesis of the amino acids is tointroduce genes that deregulate the amino acid biosynthetic pathwayssuch that the plant can no longer adequately control the levels that areproduced. This may be done by deregulating or bypassing steps in theamino acid biosynthetic pathway which are normally regulated by levelsof the amino acid end product of the pathway. Examples include theintroduction of genes that encode deregulated versions of the enzymesaspartokinase or dihydrodipicolinic acid (DHDP)-synthase for increasinglysine and threonine production, and anthranilate synthase forincreasing tryptophan production. Reduction of the catabolism of theamino acids may be accomplished by introduction of DNA sequences thatreduce or eliminate the expression of genes encoding enzymes thatcatalyse steps in the catabolic pathways such as the enzymelysine-ketoglutarate reductase.

The protein composition of the grain may be altered to improve thebalance of amino acids in a variety of ways including elevatingexpression of native proteins, decreasing expression of those with poorcomposition, changing the composition of native proteins, or introducinggenes encoding entirely new proteins possessing superior composition.DNA may be introduced that decreases the expression of members of thezein family of storage proteins. This DNA may encode ribozymes orantisense sequences directed to impairing expression of zein proteins orexpression of regulators of zein expression such as the opaque-2 geneproduct. The protein composition of the grain may be modified throughthe phenomenon of cosuppression, i.e., inhibition of expression of anendogenous gene through the expression of an identical structural geneor gene fragment introduced through transformation (Goring et al.,1991). Additionally, the introduced DNA may encode enzymes which degradeseines. The decreases in zein expression that are achieved may beaccompanied by increases in proteins with more desirable amino acidcomposition or increases in other major seed constituents such asstarch. Alternatively, a chimeric gene may be introduced that comprisesa coding-sequence for a native protein of adequate amino acidcomposition such as for one of the globulin proteins or 10 kD zein ofmaize and a promoter or other regulatory sequence designed to elevateexpression of said protein. The coding sequence of said gene may includeadditional or replacement codons for essential amino acids. Further, acoding sequence obtained from another species, or, a partially orcompletely synthetic sequence encoding a completely unique peptidesequence designed to enhance the amino acid composition of the seed maybe employed.

The introduction of genes that alter the oil content of the grain may beof value. Increases in oil content may result in increases inmetabolizable energy content and density of the seeds for uses in feedand food. The introduced genes may encode enzymes that remove or reducerate-limitations or regulated steps in fatty acid or lipid biosynthesis.Such genes may include, but are not limited to, those that encodeacetyl-CoA carboxylase, ACP-acyltransferase, beta-ketoacyl-ACP synthase,plus other well known fatty acid biosynthetic activities. Otherpossibilities are genes that encode proteins that do not possessenzymatic activity such as acyl carrier protein. Additional examplesinclude 2-acetyltransferase, oleosin pyruvate dehydrogenase complex,acetyl CoA synthetase, ATP citrate lyase, ADP-glucose pyrophosphorylaseand genes of the carnitine-CoA-acetyl-CoA shuttles. It is anticipatedthat expression of genes related to oil biosynthesis will be targeted tothe plastid, using a plastid transit peptide sequence and preferablyexpressed in the seed embryo. Genes may be introduced that alter thebalance of fatty acids present in the oil providing a more healthful ornutritive feedstuff. The introduced DNA may also encode sequences thatblock expression of enzymes involved in fatty acid biosynthesis,altering the proportions of fatty acids present in the grain such asdescribed below.

Genes may be introduced that enhance the nutritive value of the starchcomponent of the grain, for example by increasing the degree ofbranching, resulting in improved utilization of the starch in cows bydelaying its metabolism.

Besides affecting the major constituents of the grain, genes may beintroduced that affect a variety of other nutritive, processing, orother quality aspects of the grain as used for feed or food. Forexample, pigmentation of the grain may be increased or decreased.Enhancement and stability of yellow pigmentation is desirable in someanimal feeds and may be achieved by introduction of genes that result inenhanced production of xanthophylls and carotenes by eliminatingrate-limiting steps in their production. Such genes may encode alteredforms of the enzymes phytoene synthase, phytoene desaturase, or lycopenesynthase. Alternatively, unpigmented white corn is desirable forproduction of many food products and may be produced by the introductionof DNA which blocks or eliminates steps in pigment production pathways.

Feed or food comprising some cereal grains possesses insufficientquantities of vitamins and must be supplemented to provide adequatenutritive value. Introduction of genes that enhance vitamin biosynthesisin seeds may be envisioned including, for example, vitamins A, E,B.sub.12, choline, and the like. For example, maize grain also does notpossess sufficient mineral content for optimal nutritive value. Genesthat affect the accumulation or availability of compounds containingphosphorus, sulfur, calcium, manganese, zinc, and iron among otherswould be valuable. An example may be the introduction of a gene thatreduced phytic acid production or encoded the enzyme phytase whichenhances phytic acid breakdown. These genes would increase levels ofavailable phosphate in the diet, reducing the need for supplementationwith mineral phosphate.

Numerous other examples of improvement of cereals for feed and foodpurposes might be described. The improvements may not even necessarilyinvolve the grain, but may, for example, improve the value of the grainfor silage. Introduction of DNA to accomplish this might includesequences that alter lignin production such as those that result in the“brown midrib” phenotype associated with superior feed value for cattle.

In addition to direct improvements in feed or food value, genes may alsobe introduced which improve the processing of grain and improve thevalue of the products resulting from the processing. The primary methodof processing certain grains such as maize is via wetmilling. Maize maybe improved though the expression of novel genes that increase theefficiency and reduce the cost of processing such as by decreasingsteeping time.

Improving the value of wetmilling products may include altering thequantity or quality of starch, oil, corn gluten meal, or the componentsof corn gluten feed. Elevation of starch may be achieved through theidentification and elimination of rate limiting steps in starchbiosynthesis or by decreasing levels of the other components of thegrain resulting in proportional increases in starch. An example of theformer may be the introduction of genes encoding ADP-glucosepyrophosphorylase enzymes with altered regulatory activity or which areexpressed at higher level. Examples of the latter may include selectiveinhibitors of, for example, protein or oil biosynthesis expressed duringlater stages of kernel development.

The properties of starch may be beneficially altered by changing theratio of amylose to amylopectin, the size of the starch molecules, ortheir branching pattern. Through these changes a broad range ofproperties may be modified which include, but are not limited to,changes in gelatinization temperature, heat of gelatinization, clarityof films and pastes, Theological properties, and the like. To accomplishthese changes in properties, genes that encode granule-bound or solublestarch synthase activity or branching enzyme activity may be introducedalone or combination. DNA such as antisense constructs may also be usedto decrease levels of endogenous activity of these enzymes. Theintroduced genes or constructs may possess regulatory sequences thattime their expression to specific intervals in starch biosynthesis andstarch granule development. Furthermore, it may be advisable tointroduce and express genes that result in the in vivo derivatization,or other modification, of the glucose moieties of the starch molecule.The covalent attachment of any molecule may be envisioned, limited onlyby the existence of enzymes that catalyze the derivatizations and theaccessibility of appropriate substrates in the starch granule. Examplesof important derivations may include the addition of functional groupssuch as amines, carboxyls, or phosphate groups which provide sites forsubsequent in vitro derivatizations or affect starch properties throughthe introduction of ionic charges. Examples of other modifications mayinclude direct changes of the glucose units such as loss of hydroxylgroups or their oxidation to aldehyde or carboxyl groups.

Oil is another product of wetmilling of corn and other grains, the valueof which may be improved by introduction and expression of genes. Thequantity of oil that can be extracted by wetmilling may be elevated byapproaches as described for feed and food above. Oil properties may alsobe altered to improve its performance in the production and use ofcooking oil, shortenings, lubricants or other oil-derived products orimprovement of its health attributes when used in the food-relatedapplications. Novel fatty acids may also be synthesized which uponextraction can serve as starting materials for chemical syntheses. Thechanges in oil properties may be achieved by altering the type, level,or lipid arrangement of the fatty acids present in the oil. This in turnmay be accomplished by the addition of genes that encode enzymes thatcatalyze the synthesis of novel fatty acids and the lipids possessingthem or by increasing levels of native fatty acids while possiblyreducing levels of precursors. Alternatively DNA sequences may beintroduced which slow or block steps in fatty acid biosynthesisresulting in the increase in precursor fatty acid intermediates. Genesthat might be added include desaturases, epoxidases, hydratases,dehydratases, and other enzymes that catalyze reactions involving fattyacid intermediates. Representative examples of catalytic steps thatmight be blocked include the desaturations from stearic to oleic acidand oleic to linolenic acid resulting in the respective accumulations ofstearic and oleic acids.

Improvements in the other major cereal wetmilling products, gluten mealand gluten feed, may also be achieved by the introduction of genes toobtain novel plants. Representative possibilities include but are notlimited to those described above for improvement of food and feed value.

In addition it may further be considered that the plant be used for theproduction or manufacturing of useful biological compounds that wereeither not produced at all, or not produced at the same level, in theplant previously. The novel plants producing these compounds are madepossible by the introduction and expression of genes by transformationmethods. The possibilities include, but are not limited to, anybiological compound which is presently produced by any organism such asproteins, nucleic acids, primary and intermediary metabolites,carbohydrate polymers, etc. The compounds may be produced by the plant,extracted upon harvest and/or processing, and used for any presentlyrecognized useful purpose such as pharmaceuticals, fragrances,industrial enzymes to name a few.

Further possibilities to exemplify the range of grain traits orproperties potentially encoded by introduced genes in transgenic plantsinclude grain with less breakage susceptibility for export purposes orlarger grit size when processed by dry milling through introduction ofgenes that enhance gamma-zein synthesis, popcorn with improved poppingquality and expansion volume through genes that increase pericarpthickness, corn with whiter grain for food uses though introduction ofgenes that effectively block expression of enzymes involved in pigmentproduction pathways, and improved quality of alcoholic beverages orsweet corn through introduction of genes which affect flavor such as theshrunken gene (encoding sucrose synthase) for sweet corn.

7. Plant Agronomic Characteristics

Two of the factors determining where plants can be grown are the averagedaily temperature during the growing season and the length of timebetween frosts. Within the areas where it is possible to grow aparticular plant, there are varying limitations on the maximal time itis allowed to grow to maturity and be harvested. The plant to be grownin a particular area is selected for its ability to mature and dry downto harvestable moisture content within the required period of time withmaximum possible yield. Therefore, plant of varying maturities aredeveloped for different growing locations. Apart from the need to drydown sufficiently to permit harvest is the desirability of havingmaximal drying take place in the field to minimize the amount of energyrequired for additional drying post-harvest. Also the more readily thegrain can dry down, the more time there is available for growth andkernel fill. Genes that influence maturity and/or dry down can beidentified and introduced into plant lines using transformationtechniques to create new varieties adapted to different growinglocations or the same growing location but having improved yield tomoisture ratio at harvest. Expression of genes that are involved inregulation of plant development may be especially useful, e.g., theliguleless and rough sheath genes that have been identified in plants.

Genes may be introduced into plants that would improve standability andother plant growth characteristics. For example, expression of novelgenes which confer stronger stalks, improved root systems, or prevent orreduce ear droppage would be of great value to the corn farmer.Introduction and expression of genes that increase the total amount ofphotoassimilate available by, for example, increasing light distributionand/or interception would be advantageous. In addition the expression ofgenes that increase the efficiency of photosynthesis and/or the leafcanopy would further increase gains in productivity. Such approacheswould allow for increased plant populations in the field.

Delay of late season vegetative senescence would increase the flow ofassimilate into the grain and thus increase yield. Overexpression ofgenes within plants that are associated with “stay green” or theexpression of any gene that delays senescence would achieve beadvantageous. For example, a non-yellowing mutant has been identified inFestuca pratensis (Davies et al., 1990). Expression of this gene as wellas others may prevent premature breakdown of chlorophyll and thusmaintain canopy function.

8. Nutrient Utilization

The ability to utilize available nutrients and minerals may be alimiting factor in growth of many plants. It is proposed that it wouldbe possible to alter nutrient uptake, tolerate pH extremes, mobilizationthrough the plant, storage pools, and availability for metabolicactivities by the introduction of novel genes. These modifications wouldallow a plant to more efficiently utilize available nutrients. It iscontemplated that an increase in the activity of, for example, an enzymethat is normally present in the plant and involved in nutrientutilization would increase the availability of a nutrient. An example ofsuch an enzyme would be phytase. It is also contemplated that expressionof a novel gene may make a nutrient source available that was previouslynot accessible, e.g., an enzyme that releases a component of nutrientvalue from a more complex molecule, perhaps a macromolecule.

9. Male Sterility

Male sterility is useful in the production of hybrid seed. It isproposed that male sterility may be produced through expression of novelgenes. For example, it has been shown that expression of genes thatencode proteins that interfere with development of the maleinflorescence and/or gametophyte result in male sterility. Chimericribonuclease genes that express in the anthers of transgenic tobacco andoilseed rape have been demonstrated to lead to male sterility (Marianiet al, 1990).

For example, a number of mutations were discovered in maize that confercytoplasmic male sterility. One mutation in particular, referred to as Tcytoplasm, also correlates with sensitivity to Southern corn leafblight. A DNA sequence, designated TURF-13 (Levings, 1990), wasidentified that correlates with T cytoplasm. It would be possiblethrough the introduction of TURF-13 via transformation to separate malesterility from disease sensitivity. As it is necessary to be able torestore male fertility for breeding purposes and for grain production,it is proposed that genes encoding restoration of male fertility mayalso be introduced.

10. Negative Selectable Markers

Introduction of genes encoding traits that can be selected against maybe useful for eliminating undesirable linked genes. When two or moregenes are introduced together by cotransformation, the genes will belinked together on the host chromosome. For example, a gene encoding aBt gene that confers insect resistance on the plant may be introducedinto a plant together with a bar gene that is useful as a selectablemarker and confers resistance to the herbicide Ignite® on the plant.However, it may not be desirable to have an insect resistant plant thatis also resistant to the herbicide Ignite®. It is proposed that onecould also introduce an antisense bar gene that is expressed in thosetissues where one does not want expression of the bar gene, e.g., inwhole plant parts. Hence, although the bar gene is expressed and isuseful as a selectable marker, it is not useful to confer herbicideresistance on the whole plant. The bar antisense gene is a negativeselectable marker.

Negative selection is necessary in order to screen a population oftransformants for rare homologous recombinants generated through genetargeting. For example, a homologous recombinant may be identifiedthrough the inactivation of a gene that was previously expressed in thatcell. The antisense gene to neomycin phosphotransferase II (nptII) hasbeen investigated as a negative selectable marker in tobacco (Nicotianatabacum) and Arabidopsis thaliana (Xiang and Guerra, 1993). In thisexample both sense and antisense nptII genes are introduced into a plantthrough transformation and the resultant plants are sensitive to theantibiotic kanamycin. An introduced gene that integrates into the hostcell chromosome at the site of the antisense nptII gene, and inactivatesthe antisense gene, will make the plant resistant to kanamycin and otheraminoglycoside antibiotics. Therefore, rare site specific recombinantsmay be identified by screening for antibiotic resistance. Similarly, anygene, native to the plant or introduced through transformation, thatwhen inactivated confers resistance to a compound, may be useful as anegative selectable marker.

It is contemplated that negative selectable markers may also be usefulin other ways. One application is to construct transgenic lines in whichone could select for transposition to unlinked sites. In the process oftagging it is most common for the transposable element to move to agenetically linked site on the same chromosome. A selectable marker forrecovery of rare plants in which transposition has occurred to anunlinked locus would be useful. For example, the enzyme cytosinedeaminase may be useful for this purpose (Stouggard, 1993). In thepresence of this enzyme the compound 5-fluorocytosine is converted to5-fluoruracil which is toxic to plant and animal cells. If atransposable element is linked to the gene for the enzyme cytosinedeaminase, one may select for transposition to unlinked sites byselecting for transposition events in which the resultant plant is nowresistant to 5-fluorocytosine. The parental plants and plants containingtranspositions to linked sites will remain sensitive to5-fluorocytosine. Resistance to 5-fluorocytosine is due to loss of thecytosine deaminase gene through genetic segregation of the transposableelement and the cytosine deaminase gene. Other genes that encodeproteins that render the plant sensitive to a certain compound will alsobe useful in this context. For example, T-DNA gene 2 from Agrobacteriumtumefaciens encodes a protein that catalyzes the conversion ofalpha-naphthalene acetamide (NAM) to alpha-napthalene acetic acid (NAA)renders plant cells sensitive to high concentrations of NAM (Depicker etal., 1988).

It is also contemplated that negative selectable markers may be usefulin the construction of transposon tagging lines. For example, by markingan autonomous transposable element such as Ac, Master Mu, or En/Spn witha negative selectable marker, one could select for transformants inwhich the autonomous element is not stably integrated into the genome.This would be desirable, for example, when transient expression of theautonomous element is desired to activate in trans the transposition ofa defective transposable element, such as Ds, but stable integration ofthe autonomous element is not desired. The presence of the autonomouselement may not be desired in order to stabilize the defective element,i.e., prevent it from further transposing. However, it is proposed thatif stable integration of an autonomous transposable element is desiredin a plant the presence of a negative selectable marker may make itpossible to eliminate the autonomous element during the breedingprocess.

11. Non-Protein-Expressing Sequences

a. RNA-Expressing

DNA may be introduced into plants for the purpose of expressing RNAtranscripts that function to affect plant phenotype yet are nottranslated into protein. Two examples are antisense RNA and RNA withribozyme activity. Both may serve possible functions in reducing oreliminating expression of native or introduced plant genes.

Genes may be constructed or isolated, which when transcribed, produceantisense RNA that is complementary to all or part(s) of a targetedmessenger RNA(s). The antisense RNA reduces production of thepolypeptide product of the messenger RNA. The polypeptide product may beany protein encoded by the plant genome. The aforementioned genes willbe referred to as antisense genes. An antisense gene may thus beintroduced into a plant by transformation methods to produce a noveltransgenic plant with reduced expression of a selected protein ofinterest. For example, the protein may be an enzyme that catalyzes areaction in the plant. Reduction of the enzyme activity may reduce oreliminate products of the reaction which include any enzymaticallysynthesized compound in the plant such as fatty acids, amino acids,carbohydrates, nucleic acids and the like. Alternatively, the proteinmay be a storage protein, such as a zein, or a structural protein, thedecreased expression of which may lead to changes in seed amino acidcomposition or plant morphological changes respectively. Thepossibilities cited above are provided only by way of example and do notrepresent the full range of applications.

Genes may also be constructed or isolated, which when transcribedproduce RNA enzymes, or ribozymes, which can act as endoribonucleasesand catalyze the cleavage of RNA molecules with selected sequences. Thecleavage of selected messenger RNA's can result in the reducedproduction of their encoded polypeptide products. These genes may beused to prepare novel transgenic plants which possess them. Thetransgenic plants may possess reduced levels of polypeptides includingbut not limited to the polypeptides cited above that may be affected byantisense RNA.

It is also possible that genes may be introduced to produce noveltransgenic plants which have reduced expression of a native gene productby a mechanism of cosuppression. It has been demonstrated in tobacco,tomato, and petunia (Goring et al, 1991; Smith et al., 1990; Napoli etal., 1990; van der Krol et al., 1990) that expression of the sensetranscript of a native gene will reduce or eliminate expression of thenative gene in a manner similar to that observed for antisense genes.The introduced gene may encode all or part of the targeted nativeprotein but its translation may not be required for reduction of levelsof that native protein.

b. Non-RNA-Expressing

For example, DNA elements including those of transposable elements suchas Ds, Ac, or Mu, may be inserted into a gene and cause mutations. TheseDNA elements may be inserted in order to inactivate (or activate) a geneand thereby “tag” a particular trait. In this instance the transposableelement does not cause instability of the tagged mutation, because theutility of the element does not depend on its ability to move in thegenome. Once a desired trait is tagged, the introduced DNA sequence maybe used to clone the corresponding gene, e.g., using the introduced DNAsequence as a PCR primer together with PCR gene cloning techniques(Shapiro, 1983; Dellaporta et al., 1988). Once identified, the entiregene(s) for the particular trait, including control or regulatoryregions where desired may be isolated, cloned and manipulated asdesired. The utility of DNA elements introduced into an organism forpurposed of gene tagging is independent of the DNA sequence and does notdepend on any biological activity of the DNA sequence, i.e.,transcription into RNA or translation into protein. The sole function ofthe DNA element is to disrupt the DNA sequence of a gene.

It is contemplated that unexpressed DNA sequences, including novelsynthetic sequences could be introduced into cells as proprietary“labels” of those cells and plants and seeds thereof. It would not benecessary for a label DNA element to disrupt the function of a geneendogenous to the host organism, as the sole function of this DNA wouldbe to identify the origin of the organism. For example, one couldintroduce a unique DNA sequence into a plant and this DNA element wouldidentify all cells, plants, and progeny of these cells as having arisenfrom that labeled source. It is proposed that inclusion of label DNAswould enable one to distinguish proprietary germplasm or germplasmderived from such, from unlabelled germplasm.

Another possible element which may be introduced is a matrix attachmentregion element (MAR), such as the chicken lysozyme A element (Stief etal., 1989), which can be positioned around an expressible gene ofinterest to effect an increase in overall expression of the gene anddiminish position dependent effects upon incorporation into the plantgenome (Stief et al., 1989; Phi-Van et al., 1990).

III. TRANSFORMED (TRANSGENIC) PLANTS OF THE INVENTION AND METHODS OFPREPARATION

Plant species may be transformed with the DNA construct of the presentinvention by the DNA-mediated transformation of plant cell protoplastsand subsequent regeneration of the plant from the transformedprotoplasts in accordance with procedures well known in the art.

Any plant tissue capable of subsequent clonal propagation, whether byorganogenesis or embryogenesis, may be transformed with a vector of thepresent invention. The term “organogenesis,” as used herein, means aprocess by which shoots and roots are developed sequentially frommeristematic centers; the term “embryogenesis,” as used herein, means aprocess by which shoots and roots develop together in a concertedfashion (not sequentially), whether from somatic cells or gametes. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, megagametophytes, callus tissue,existing meristematic tissue (e.g., apical meristems, axillary buds, androot meristems), and induced meristem tissue (e.g., cotyledon meristemand ultilane meristem).

Plants of the present invention may take a variety of forms. The plantsmay be chimeras of transformed cells and non-transformed cells; theplants may be clonal transformants (e.g., all cells transformed tocontain the expression cassette); the plants may comprise grafts oftransformed and untransformed tissues (e.g., a transformed root stockgrafted to an untransformed scion in citrus species). The transformedplants may be propagated by a variety of means, such as by clonalpropagation or classical breeding techniques. For example, firstgeneration (or T1) transformed plants may be selfed to give homozygoussecond generation (or T2) transformed plants, and the T2 plants furtherpropagated through classical breeding techniques. A dominant selectablemarker (such as npt II) can be associated with the expression cassetteto assist in breeding.

Thus, the present invention provides a transformed (transgenic) plantcell, in planta or ex planta, including a transformed plastid or otherorganelle, e.g., nucleus, mitochondria or chloroplast. The presentinvention may be used for transformation of any plant species,including, but not limited to, cells from corn (Zea mays), Brassica sp.(e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea ultilane), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, duckweed(Lemna), barley, vegetables, ornamentals, and conifers.

Duckweed (Lemna, see WO 00/07210) includes members of the familyLemnaceae. There are known four genera and 34 species of duckweed asfollows: genus Lemna (L. aequinoctialis, L. disperma, L. ecuadoriensis,L. gibba, L. japonica, L. minor, L. miniscula, L. obscura, L.perpusilla, L. tenera, L. trisulca, L. turionifera, L. valdiviana);genus Spirodela (S. intermedia, S. polyrrhiza, S. punctata); genusWoffia (Wa. Angusta, Wa. Arrhiza, Wa. Australina, Wa. Borealis, Wa.Brasiliensis, Wa. Columbiana, Wa. Elongata, Wa. Globosa, Wa.Microscopica, Wa. Neglecta) and genus Wofiella (W1. ultila, W1.ultilanen, W1. gladiata, W1. ultila, W1. lingulata, W1. repunda, W1.rotunda, and W1. neotropica). Any other genera or species of Lemnaceae,if they exist, are also aspects of the present invention. Lemna gibba,Lemna minor, and Lemna miniscula are preferred, with Lemna minor andLemna miniscula being most preferred. Lemna species can be classifiedusing the taxonomic scheme described by Landolt, BiosystematicInvestigation on the Family of Duckweeds: The family of Lemnaceae—AMonograph Study. Geobatanischen Institut ETH, Stiftung Rubel, Zurich(1986)).

Vegetables within the scope of the invention include tomatoes(Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans(Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrusspp.), and members of the genus Cucumis such as cucumber (C. sativus),cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentalsinclude azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipaspp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation(Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), andchrysanthemum. Conifers that may be employed in practicing the presentinvention include, for example, pines such as loblolly pine (Pinustaeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa),lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata),Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga ultilane);Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firssuch as silver fir (Abies amabilis) and balsam fir (Abies balsamea); andcedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). Leguminous plants include beans and peas.Beans include guar, locust bean, fenugreek, soybean, garden beans,cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc. Legumesinclude, but are not limited to, Arachis, e.g., peanuts, Vicia, e.g.,crown vetch, hairy vetch, adzuki bean, mung bean, and chickpea, Lupinus,e.g., lupine, trifolium, Phaseolus, e.g., common bean and lima bean,Pisum, e.g., field bean, Melilotus, e.g., clover, Medicago, e.g.,alfalfa, Lotus, e.g., trefoil, lens, e.g., lentil, and false indigo.Preferred forage and turf grass for use in the methods of the inventioninclude alfalfa, orchard grass, tall fescue, perennial ryegrass,creeping bent grass, and redtop.

Other plants within the scope of the invention include Acacia, aneth,artichoke, arugula, blackberry, canola, cilantro, clementines, escarole,eucalyptus, fennel, grapefruit, honey dew, jicama, kiwifruit, lemon,lime, mushroom, nut, okra, orange, parsley, persimmon, plantain,pomegranate, poplar, radiata pine, radicchio, Southern pine, sweetgum,tangerine, triticale, vine, yams, apple, pear, quince, cherry, apricot,melon, hemp, buckwheat, grape, raspberry, chenopodium, blueberry,nectarine, peach, plum, strawberry, watermelon, eggplant, pepper,cauliflower, Brassica, e.g., broccoli, cabbage, ultilan sprouts, onion,carrot, leek, beet, broad bean, celery, radish, pumpkin, endive, gourd,garlic, snapbean, spinach, squash, turnip, ultilane, and zucchini.

Ornamental plants within the scope of the invention include impatiens,Begonia, Pelargonium, Viola, Cyclamen, Verbena, Vinca, Tagetes, Primula,Saint Paulia, Agertum, Amaranthus, Antihirrhinum, Aquilegia, Cineraria,Clover, Cosmo, Cowpea, Dahlia, Datura, Delphinium, Gerbera, Gladiolus,Gloxinia, Hippeastrum, Mesembryanthemum, Salpiglossos, and Zinnia. Otherplants within the scope of the invention are shown in Table 1 (above).

Preferably, transgenic plants of the present invention are crop plantsand in particular cereals (for example, corn, alfalfa, sunflower, rice,Brassica, canola, soybean, barley, soybean, sugarbeet, cotton,safflower, peanut, sorghum, wheat, millet, tobacco, etc.), and even morepreferably corn, rice and soybean.

Transformation of plants can be undertaken with a single DNA molecule ormultiple DNA molecules (i.e., co-transformation), and both thesetechniques are suitable for use with the expression cassettes of thepresent invention. Numerous transformation vectors are available forplant transformation, and the expression cassettes of this invention canbe used in conjunction with any such vectors. The selection of vectorwill depend upon the preferred transformation technique and the targetspecies for transformation.

A variety of techniques are available and known to those skilled in theart for introduction of constructs into a plant cell host. Thesetechniques generally include transformation with DNA employing A.tumefaciens or A. rhizogenes as the transforming agent, liposomes, PEGprecipitation, electroporation, DNA injection, direct DNA uptake,microprojectile bombardment, particle acceleration, and the like (See,for example, EP 295959 and EP 138341) (see below). However, cells otherthan plant cells may be transformed with the expression cassettes of theinvention. The general descriptions of plant expression vectors andreporter genes, and Agrobacterium and Agrobacterium-mediated genetransfer, can be found in Gruber et al. (1993).

Expression vectors containing genomic or synthetic fragments can beintroduced into protoplasts or into intact tissues or isolated cells.Preferably expression vectors are introduced into intact tissue. Generalmethods of culturing plant tissues are provided for example by Maki etal., (1993); and by Phillips et al. (1988). Preferably, expressionvectors are introduced into maize or other plant tissues using a directgene transfer method such as microprojectile-mediated delivery, DNAinjection, electroporation and the like. More preferably expressionvectors are introduced into plant tissues using the microprojectilemedia delivery with the biolistic device. See, for example, Tomes et al.(1995). The vectors of the invention can not only be used for expressionof structural genes but may also be used in exon-trap cloning, orpromoter trap procedures to detect differential gene expression invarieties of tissues, (Lindsey et al., 1993; Auch & Reth et al.).

It is particularly preferred to use the binary type vectors of Ti and Riplasmids of Agrobacterium spp. Ti-derived vectors transform a widevariety of higher plants, including monocotyledonous and dicotyledonousplants, such as soybean, cotton, rape, tobacco, and rice (Pacciotti etal., 1985: Byrne et al., 1987; Sukhapinda et al., 1987; Park et al.,1985: Hiei et al., 1994). The use of T-DNA to transform plant cells hasreceived extensive study and is amply described (EP 120516; Hoekema,1985; Knauf, et al., 1983; and An et al., 1985). For introduction intoplants, the chimeric genes of the invention can be inserted into binaryvectors as described in the examples.

Other transformation methods are available to those skilled in the art,such as direct uptake of foreign DNA constructs (see EP 295959),techniques of electroporation (Fromm et al., 1986) or high velocityballistic bombardment with metal particles coated with the nucleic acidconstructs (Kline et al., 1987, and U.S. Pat. No. 4,945,050). Oncetransformed, the cells can be regenerated by those skilled in the art.Of particular relevance are the recently described methods to transformforeign genes into commercially important crops, such as rapeseed (DeBlock et al., 1989), sunflower (Everett et al., 1987), soybean (McCabeet al., 1988; Hinchee et al., 1988; Chee et al., 1989; Christou et al.,1989; EP 301749), rice (Hiei et al., 1994), and corn (Gordon Kamm etal., 1990; Fromm et al., 1990).

Those skilled in the art will appreciate that the choice of method mightdepend on the type of plant, i.e., monocotyledonous or dicotyledonous,targeted for transformation. Suitable methods of transforming plantcells include, but are not limited to, microinjection (Crossway et al.,1986), electroporation (Riggs et al., 1986), Agrobacterium-mediatedtransformation (Hinchee et al., 1988), direct gene transfer (Paszkowskiet al., 1984), and ballistic particle acceleration using devicesavailable from Agracetus, Inc., Madison, Wis. And BioRad, Hercules,Calif. (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; andMcCabe et al., 1988). Also see, Weissinger et al., 1988; Sanford et al.,1987 (onion); Christou et al., 1988 (soybean); McCabe et al., 1988(soybean); Datta et al., 1990 (rice); Klein et al., 1988 (maize); Kleinet al., 1988 (maize); Klein et al., 1988 (maize); Fromm et al., 1990(maize); and Gordon-Kamm et al., 1990 (maize); Svab et al., 1990(tobacco chloroplast); Koziel et al., 1993 (maize); Shimamoto et al.,1989 (rice); Christou et al., 1991 (rice); European Patent ApplicationEP 0 332 581 (orchardgrass and other Pooideae); Vasil et al., 1993(wheat); Weeks et al., 1993 (wheat). In one embodiment, the protoplasttransformation method for maize is employed (European Patent ApplicationEP 0 292 435, U.S. Pat. No. 5,350,689).

In another embodiment, a nucleotide sequence of the present invention isdirectly transformed into the plastid genome. Plastid transformationtechnology is extensively described in U.S. Pat. Nos. 5,451,513,5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and inMcBride et al., 1994. The basic technique for chloroplast transformationinvolves introducing regions of cloned plastid DNA flanking a selectablemarker together with the gene of interest into a suitable target tissue,e.g., using biolistics or protoplast transformation (e.g., calciumchloride or PEG mediated transformation). The 1 to 1.5 kb flankingregions, termed targeting sequences, facilitate orthologousrecombination with the plastid genome and thus allow the replacement ormodification of specific regions of the plastome. Initially, pointmutations in the chloroplast 16S rRNA and rps12 genes conferringresistance to spectinomycin and/or streptomycin are utilized asselectable markers for transformation (Svab et al., 1990; Staub et al.,1992). This resulted in stable homoplasmic transformants at a frequencyof approximately one per 100 bombardments of target leaves. The presenceof cloning sites between these markers allowed creation of a plastidtargeting vector for introduction of foreign genes (Staub et al., 1993).Substantial increases in transformation frequency are obtained byreplacement of the recessive rRNA or r-protein antibiotic resistancegenes with a dominant selectable marker, the bacterial aadA geneencoding the spectinomycin-detoxifying enzymeaminoglycoside-3′-adenyltransferase (Svab et al., 1993). Otherselectable markers useful for plastid transformation are known in theart and encompassed within the scope of the invention. Typically,approximately 15-20 cell division cycles following transformation arerequired to reach a homoplastidic state. Plastid expression, in whichgenes are inserted by orthologous recombination into all of the severalthousand copies of the circular plastid genome present in each plantcell, takes advantage of the enormous copy number advantage overnuclear-expressed genes to permit expression levels that can readilyexceed 10% of the total soluble plant protein. In a preferredembodiment, a nucleotide sequence of the present invention is insertedinto a plastid targeting vector and transformed into the plastid genomeof a desired plant host. Plants homoplastic for plastid genomescontaining a nucleotide sequence of the present invention are obtained,and are preferentially capable of high expression of the nucleotidesequence.

Agrobacterium tumefaciens cells containing a vector comprising anexpression cassette of the present invention, wherein the vectorcomprises a Ti plasmid, are useful in methods of making transformedplants. Plant cells are infected with an Agrobacterium tumefaciens asdescribed above to produce a transformed plant cell, and then a plant isregenerated from the transformed plant cell. Numerous Agrobacteriumvector systems useful in carrying out the present invention are known.

For example, vectors are available for transformation usingAgrobacterium tumefaciens. These typically carry at least one T-DNAborder sequence and include vectors such as pBIN19 (Bevan, 1984). In onepreferred embodiment, the expression cassettes of the present inventionmay be inserted into either of the binary vectors pCIB200 and pCIB2001for use with Agrobacterium. These vector cassettes forAgrobacterium-mediated transformation wear constructed in the followingmanner. PTJS75kan was created by Narl digestion of pTJS75 (Schmidhauser& Helinski, 1985) allowing excision of the tetracycline-resistance gene,followed by insertion of an Accl fragment from pUC4K carrying an NPTII(Messing & Vierra, 1982; Bevan et al., 1983; McBride et al., 1990). XhoIlinkers were ligated to the EcoRV fragment of pCIB7 which contains theleft and right T-DNA borders, a plant selectable nos/nptII chimeric geneand the pUC polylinker (Rothstein et al., 1987), and the XhoI-digestedfragment was cloned into SalI-digested pTJS75kan to create pCIB200 (seealso EP 0 332 104, example 19). PCIB200 contains the following uniquepolylinker restriction sites: EcoRI, SstI, KpnI, BglII, XbaI, and SalI.The plasmid pCIB2001 is a derivative of pCIB200 which was created by theinsertion into the polylinker of additional restriction sites. Uniquerestriction sites in the polylinker of pCIB2001 are EcoRI, SstI, KpnI,BglII, XbaI, SalI, MluI, BclI, AvrII, ApaI, HpaI, and StuI. PCIB2001, inaddition to containing these unique restriction sites also has plant andbacterial kanamycin selection, left and right T-DNA borders forAgrobacterium-mediated transformation, the RK2-derived trfA function formobilization between E. coli and other hosts, and the OriT and OriVfunctions also from RK2. The pCIB2001 polylinker is suitable for thecloning of plant expression cassettes containing their own regulatorysignals.

An additional vector useful for Agrobacterium-mediated transformation isthe binary vector pCIB 10, which contains a gene encoding kanamycinresistance for selection in plants, T-DNA right and left bordersequences and incorporates sequences from the wide host-range plasmidpRK252 allowing it to replicate in both E. coli and Agrobacterium. Itsconstruction is described by Rothstein et al., 1987. Various derivativesof pCIB 10 have been constructed which incorporate the gene forhygromycin B phosphotransferase described by Gritz et al., 1983. Thesederivatives enable selection of transgenic plant cells on hygromycinonly (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717).

Methods using either a form of direct gene transfer orAgrobacterium-mediated transfer usually, but not necessarily, areundertaken with a selectable marker which may provide resistance to anantibiotic (e.g., kanamycin, hygromycin or methotrexate) or a herbicide(e.g., phosphinothricin). The choice of selectable marker for planttransformation is not, however, critical to the invention.

For certain plant species, different antibiotic or herbicide selectionmarkers may be preferred. $election markers used routinely intransformation include the nptII gene which confers resistance tokanamycin and related antibiotics (Messing & Vierra, 1982; Bevan et al.,1983), the bar gene which confers resistance to the herbicidephosphinothricin (White et al., 1990, Spencer et al., 1990), the hphgene which confers resistance to the antibiotic hygromycin (Blochinger &Diggelmann), and the dhfr gene, which confers resistance to methotrexate(Bourouis et al., 1983).

One such vector useful for direct gene transfer techniques incombination with selection by the herbicide Basta (or phosphinothricin)is pCIB3064. This vector is based on the plasmid pCIB246, whichcomprises the CaMV 35S promoter in operational fusion to the E. coli GUSgene and the CaMV 35S transcriptional terminator and is described in thePCT published application WO 93/07278, herein incorporated by reference.One gene useful for conferring resistance to phosphinothricin is the bargene from Streptomyces viridochromogenes (Thompson et al., 1987). Thisvector is suitable for the cloning of plant expression cassettescontaining their own regulatory signals.

An additional transformation vector is pSOG35 which utilizes the E. coligene dihydrofolate reductase (DHFR) as a selectable marker conferringresistance to methotrexate. PCR was used to amplify the 35S promoter(about 800 bp), intron 6 from the maize Adh1 gene (about 550 bp) and 18bp of the GUS untranslated leader sequence from pSOG10. A 250 bpfragment encoding the E. coli dihydrofolate reductase type II gene wasalso amplified by PCR and these two PCR fragments were assembled with aSacI-PstI fragment from pBI221 (Clontech) which comprised the pUC19vector backbone and the nopaline synthase terminator. Assembly of thesefragments generated pSOG19 which contains the 35S promoter in fusionwith the intron 6 sequence, the GUS leader, the DHFR gene and thenopaline synthase terminator. Replacement of the GUS leader in pSOG19with the leader sequence from Maize Chlorotic Mottle Virus check (MCMV)generated the vector pSOG35. pSOG19 and pSOG35 carry the pUC-derivedgene for ampicillin resistance and have HindIII, SphI, PstI and EcoRIsites available for the cloning of foreign sequences.

IV. PRODUCTION AND CHARACTERIZATION OF STABLY TRANSFORMED PLANTS

Transgenic plant cells are then placed in an appropriate selectivemedium for selection of transgenic cells which are then grown to callus.Shoots are grown from callus and plantlets generated from the shoot bygrowing in rooting medium. The various constructs normally will bejoined to a marker for selection in plant cells. Conveniently, themarker may be resistance to a biocide (particularly an antibiotic, suchas kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide,or the like). The particular marker used will allow for selection oftransformed cells as compared to cells lacking the DNA which has beenintroduced. Components of DNA constructs including transcriptioncassettes of this invention may be prepared from sequences which arenative (endogenous) or foreign (exogenous) to the host. By “foreign” itis meant that the sequence is not found in the wild-type host into whichthe construct is introduced. Heterologous constructs will contain atleast one region which is not native to the gene from which thetranscription-initiation-region is derived.

To confirm the presence of the transgenes in transgenic cells andplants, a variety of assays may be performed. Such assays include, forexample, “molecular biological” assays well known to those of skill inthe art, such as Southern and Northern blotting, in situ hybridizationand nucleic acid-based amplification methods such as PCR or RT-PCR;“biochemical” assays, such as detecting the presence of a proteinproduct, e.g., by immunological means (ELISAs and Western blots) or byenzymatic function; plant part assays, such as leaf or root assays; andalso, by analyzing the phenotype of the whole regenerated plant, e.g.,for disease or pest resistance.

DNA may be isolated from cell lines or any plant parts to determine thepresence of the preselected nucleic acid segment through the use oftechniques well known to those skilled in the art. Note that intactsequences will not always be present, presumably due to rearrangement ordeletion of sequences in the cell.

The presence of nucleic acid elements introduced through the methods ofthis invention may be determined by polymerase chain reaction (PCR).Using this technique discreet fragments of nucleic acid are amplifiedand detected by gel electrophoresis. This type of analysis permits oneto determine whether a preselected nucleic acid segment is present in astable transformant, but does not prove integration of the introducedpreselected nucleic acid segment into the host cell genome. In addition,it is not possible using PCR techniques to determine whethertransformants have exogenous genes introduced into different sites inthe genome, i.e., whether transformants are of independent origin. It iscontemplated that using PCR techniques it would be possible to clonefragments of the host genomic DNA adjacent to an introduced preselectedDNA segment.

Positive proof of DNA integration into the host genome and theindependent identities of transformants may be determined using thetechnique of Southern hybridization. Using this technique specific DNAsequences that were introduced into the host genome and flanking hostDNA sequences can be identified. Hence the Southern hybridizationpattern of a given transformant serves as an identifying characteristicof that transformant. In addition it is possible through Southernhybridization to demonstrate the presence of introduced preselected DNAsegments in high molecular weight DNA, i.e., confirm that the introducedpreselected DNA segment has been integrated into the host cell genome.The technique of Southern hybridization provides information that isobtained using PCR, e.g., the presence of a preselected DNA segment, butalso demonstrates integration into the genome and characterizes eachindividual transformant.

It is contemplated that using the techniques of dot or slot blothybridization which are modifications of Southern hybridizationtechniques one could obtain the same information that is derived fromPCR, e.g., the presence of a preselected DNA segment.

Both PCR and Southern hybridization techniques can be used todemonstrate transmission of a preselected DNA segment to progeny. Inmost instances the characteristic Southern hybridization pattern for agiven transformant will segregate in progeny as one or more Mendeliangenes (Spencer et al., 1992); Laursen et al., 1994) indicating stableinheritance of the gene. The nonchimeric nature of the callus and theparental transformants (R₀) was suggested by germine transmission andthe identical Southern blot hybridization patterns and intensities ofthe transforming DNA in callus, R₀ plants and R₁ progeny that segregatedfor the transformed gene.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA may only be expressed in particular cells ortissue types and hence it will be necessary to prepare RNA for analysisfrom these tissues. PCR techniques may also be used for detection andquantitation of RNA produced from introduced preselected DNA segments.In this application of PCR it is first necessary to reverse transcribeRNA into DNA, using enzymes such as reverse transcriptase, and thenthrough the use of conventional PCR techniques amplify the DNA. In mostinstances PCR techniques, while useful, will not demonstrate integrityof the RNA product. Further information about the nature of the RNAproduct may be obtained by Northern blotting. This technique willdemonstrate the presence of an RNA species and give information aboutthe integrity of that RNA. The presence or absence of an RNA species canalso be determined using dot or slot blot Northern hybridizations. Thesetechniques are modifications of Northern blotting and will onlydemonstrate the presence or absence of an RNA species.

While Southern blotting and PCR may be used to detect the preselectedDNA segment in question, they do not provide information as to whetherthe preselected DNA segment is being expressed. Expression may beevaluated by specifically identifying the protein products of theintroduced preselected DNA segments or evaluating the phenotypic changesbrought about by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focusing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity such as Western blotting in which antibodies are used tolocate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may beadditionally used.

Assay procedures may also be used to identify the expression of proteinsby their functionality, especially the ability of enzymes to catalyzespecific chemical reactions involving specific substrates and products.These reactions may be followed by providing and quantifying the loss ofsubstrates or the generation of products of the reactions by physical orchemical procedures. Examples are as varied as the enzyme to beanalyzed.

Very frequently the expression of a gene product is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms including but not limited to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant. Morphological changes may include greater stature or thickerstalks. Most often changes in response of plants or plant parts toimposed treatments are evaluated under carefully controlled conditionstermed bioassays.

V. USES OF TRANSGENIC PLANTS

Once an expression cassette of the invention has been transformed into aparticular plant species, it may be propagated in that species or movedinto other varieties of the same species, particularly includingcommercial varieties, using traditional breeding techniques.Particularly preferred plants of the invention include the agronomicallyimportant crops listed above. The genetic properties engineered into thetransgenic seeds and plants described above are passed on by sexualreproduction and can thus be maintained and propagated in progenyplants. The present invention also relates to a transgenic plant cell,tissue, organ, seed or plant part obtained from the transgenic plant.Also included within the invention are transgenic descendants of theplant as well as transgenic plant cells, tissues, organs, seeds andplant parts obtained from the descendants.

Preferably, the expression cassette in the transgenic plant is sexuallytransmitted. In one preferred embodiment, the coding sequence issexually transmitted through a complete normal sexual cycle of the R₀plant to the R₁ generation. Additionally preferred, the expressioncassette is expressed in the cells, tissues, seeds or plant of atransgenic plant in an amount that is different than the amount in thecells, tissues, seeds or plant of a plant which only differs in that theexpression cassette is absent.

The transgenic plants produced herein are thus expected to be useful fora variety of commercial and research purposes. Transgenic plants can becreated for use in traditional agriculture to possess traits beneficialto the grower (e.g., agronomic traits such as resistance to waterdeficit, pest resistance, herbicide resistance or increased yield),beneficial to the consumer of the grain harvested from the plant (e.g.,improved nutritive content in human food or animal feed; increasedvitamin, amino acid, and antioxidant content; the production ofantibodies (passive immunization) and nutriceuticals), or beneficial tothe food processor (e.g., improved processing traits). In such uses, theplants are generally grown for the use of their grain in human or animalfoods. Additionally, the use of root-specific promoters in transgenicplants can provide beneficial traits that are localized in theconsumable (by animals and humans) roots of plants such as carrots,parsnips, and beets. However, other parts of the plants, includingstalks, husks, vegetative parts, and the like, may also have utility,including use as part of animal silage or for ornamental purposes.Often, chemical constituents (e.g., oils or starches) of maize and othercrops are extracted for foods or industrial use and transgenic plantsmay be created which have enhanced or modified levels of suchcomponents.

Transgenic plants may also find use in the commercial manufacture ofproteins or other molecules, where the molecule of interest is extractedor purified from plant parts, seeds, and the like. Cells or tissue fromthe plants may also be cultured, grown in vitro, or fermented tomanufacture such molecules.

The transgenic plants may also be used in commercial breeding programs,or may be crossed or bred to plants of related crop species.Improvements encoded by the expression cassette may be transferred,e.g., from maize cells to cells of other species, e.g., by protoplastfusion.

The transgenic plants may have many uses in research or breeding,including creation of new mutant plants through insertional mutagenesis,in order to identify beneficial mutants that might later be created bytraditional mutation and selection. An example would be the introductionof a recombinant DNA sequence encoding a transposable element that maybe used for generating genetic variation. The methods of the inventionmay also be used to create plants having unique “signature sequences” orother marker sequences which can be used to identify proprietary linesor varieties.

Thus, the transgenic plants and seeds according to the invention can beused in plant breeding which aims at the development of plants withimproved properties conferred by the expression cassette, such astolerance of drought, disease, or other stresses. The various breedingsteps are characterized by well-defined human intervention such asselecting the lines to be crossed, directing pollination of the parentallines, or selecting appropriate descendant plants. Depending on thedesired properties different breeding measures are taken. The relevanttechniques are well known in the art and include but are not limited tohybridization, inbreeding, backcross breeding, multiline breeding,variety blend, interspecific hybridization, aneuploid techniques, etc.Hybridization techniques also include the sterilization of plants toyield male or female sterile plants by mechanical, chemical orbiochemical means. Cross pollination of a male sterile plant with pollenof a different line assures that the genome of the male sterile butfemale fertile plant will uniformly obtain properties of both parentallines. Thus, the transgenic seeds and plants according to the inventioncan be used for the breeding of improved plant lines which for exampleincrease the effectiveness of conventional methods such as herbicide orpesticide treatment or allow to dispense with said methods due to theirmodified genetic properties. Alternatively new crops with improvedstress tolerance can be obtained which, due to their optimized genetic“equipment”, yield harvested product of better quality than productswhich were not able to tolerate comparable adverse developmentalconditions.

VI. A COMPUTER READABLE MEDIUM

The invention also provides a computer readable medium having storedthereon a data structure containing nucleic acid sequences having e.g.,at least 70% sequence identity to a nucleic acid sequence selected fromthose listed in SEQ ID Nos: 1-26, as well as complementary, ortholog andvariant sequences thereof. Storage and use of nucleic acid sequences ona computer readable medium is well known in the art. (See for exampleU.S. Pat. Nos. 6,023,659; 5,867,402; 5,795,716) Examples of such mediuminclude, but are not limited to, magnetic tape, optical disk, CD-ROM,random access memory, volatile memory, non-volatile memory and bubblememory. Accordingly, the nucleic acid sequences contained on thecomputer readable medium may be compared through use of a module thatreceives the sequence information and compares it to other sequenceinformation. Examples of other sequences to which the nucleic acidsequences of the invention may be compared include those maintained bythe National Center for Biotechnology Information (NCBI) and the SwissProtein Data Bank. A computer is an example of such a module that canread and compare nucleic acid sequence information. Accordingly, theinvention also provides the method of comparing a nucleic acid sequenceof the invention to another sequence. For example, a sequence of theinvention may be submitted to the NCBI for a Blast search as describedherein where the sequence is compared to sequence information containedwithin the NCBI database and a comparison is returned. The inventionalso provides nucleic acid sequence information in a computer readablemedium that allows the encoded polypeptide to be optimized for a desiredproperty. Examples of such properties include, but are not limited to,increased or decreased: thermal stability, chemical stability,hydrophylicity, hydrophobicity, and the like. Methods for the use ofcomputers to model polypeptides and polynucleotides having alteredactivities are well known in the art and have been reviewed. (Lesyng etal., 1993; Surles et al., 1994; Koehl et al., 1996; Rossi et al., 2001).

The invention will be further described by the following non-limitingexamples.

Example 1 GENECHIP® Standard Protocol Quantitation of Total RNA

Total RNA from plant tissue is extracted and quantified.

-   -   1. Quantify total RNA using GeneQuant 1OD₂₆₀=40 mg RNA/ml;        A₂₆₀/A₂₈₀=1.9 to about 2.1    -   2. Run gel to check the integrity and purity of the extracted        RNA        Synthesis of Double-Stranded cDNA

Gibco/BRL SuperScript Choice System for cDNA Synthesis (Cat#1B090-019)was employed to prepare cDNAs. T7-(dT)₂₄ oligonucleotides were preparedand purified by HPLC. (5′-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)₂₄-3′; SEQ ID NO:27).

Step 1. Primer Hybridization:

-   -   Incubate at 70° C. for 10 minutes    -   Quick spin and put on ice briefly

Step 2. Temperature Adjustment:

-   -   Incubate at 42° C. for 2 minutes

Step 3. First Strand Synthesis:

-   -   DEPC-water-1 μl    -   RNA (10 μg final)-10 μl    -   T7=(dT)₂₄ Primer (100 pmol final)-1 μl pmol    -   5×1^(st) strand cDNA buffer-4 μl    -   0.1M DTT (10 mM final)-2 μl    -   10 mM dNTP mix (500 μM final)-1 μl    -   Superscript II RT 200 U/μl-1 μl    -   Total of 20 μl

Mix Well

Incubate at 42° C. for 1 hour

Step 4. Second Strand Synthesis:

-   -   Place reactions on ice, quick spin    -   DEPC-water-91 μl    -   5×2^(nd) strand cDNA buffer-30 μl    -   10 mM dNTP mix (250 mM final)-3 μl    -   E. coli DNA ligase (10 U/μl)-1 μl    -   E. coli DNA polymerase 1-10 U/μl-4 μl    -   RnaseH 2 U/μl-1 μl    -   T4 DNA polymerase 5 U/μl-2 μl    -   0.5 M EDTA (0.5 M final)-10 μl    -   Total 162 μl    -   Mix/spin down/incubate 16° C. for 2 hours

Step 5. Completing the Reaction:

Incubate at 16° C. for 5 minutes

Purification of Double Stranded cDNA

1. Centrifuge PLG (Phase Lock Gel, Eppendorf 5 Prime Inc., pl-188233) at14,000×, transfer 162 μl of cDNA to PLG

2. Add 162 μl of Phenol:Chloroform:Isoamyl alcohol (pH 8.0), centrifuge2 minutes

3. Transfer the supernatant to a fresh 1.5 ml tube, add

Glycogen (5 mg/ml)  2 μl 0.5 M NH₄OAC (0.75 × Vol) 120 μl ETOH (2.5xVol, −20° C.) 400 μl

4. Mix well and centrifuge at 14,000× for 20 minutes

5. Remove supernatant, add 0.5 ml 80% EtOH (−20° C.)

6. Centrifuge for 5 minutes, air dry or by speed vac for 5-10 minutes

7. Add 44 μl DEPC H₂O

Analyze of Quantity and Size Distribution of cDNARun a gel using 1 μl of the double-stranded synthesis productSynthesis of Biotinylated cRNA

(use Enzo BioArray High Yield RNA Transcript Labeling Kit Cat#900182)

Purified cDNA 22 μl  10X Hy buffer 4 μl 10X biotin ribonucleotides 4 μl10X DTT 4 μl 10X Rnase inhibitor mix 4 μl 20X T7 RNA polymerase 2 μlTotal 40 μl 

Centrifuge 5 seconds, and incubate for 4 hours at 37° C.

Gently mix every 30-45 minutes

Purification and Quantification of cRNA

(use Qiagen Rneasy Mini kit Cat# 74103)

cRNA 40 μl DEPC H₂O 60 μl RLT buffer 350 μl mix by vortexing EtOH 250 μlmix by pipetting Total 700 μl Wait 1 minute or more for the RNA to stickCentrifuge at 2000 rpm for 5 minutes RPE buffer 500 μl Centrifuge at10,000 rpm for 1 minute RPE buffer 500 μl Centrifuge at 10,000 rpm for 1minute Centrifuge at 10,000 rpm for 1 minute to dry the column DEPC H₂O30 μl Wait for 1 minute, then elute cRNA from by centrifugation, 10 K 1minute DEPC H₂O 30 μlRepeat previous stepDetermine concentration and dilute to 1 μg/μl concentrationFragmentation of cRNA

cRNA (1 μg/μl) 15 μl 5X Fragmentation Buffer* 6 μl DEPC H₂O 9 μl 30 μl*5x Fragmentation Buffer 1M Tris (pH8.1) 4.0 ml MgOAc 0.64 g KOAC 0.98 gDEPC H₂O Total 20 ml Filter Sterilize

Array Wash and Staining Stringent Wash Buffer** Non-Stringent WashBuffer*** SAPE Stain****

Antibody Stain*****

**Stringent Buffer: 12×MES 83.3 ml, 5 M NaCl 5.2 ml, 10% Tween 1.0 ml,H₂O 910 ml, Filter Sterilize ***Non-Stringent Buffer: 20×SSPE 300 ml,10% Tween 1.0 ml, H₂O 698 ml, Filter Sterilize, Antifoam 1.0.

****SAPE stain: 2× Stain Buffer 600 III, BSA 48 μl, SAPE 12 μl, H₂O 540μl.

*****Antibody Stain: 2× Stain Buffer 300 μl, H.sub.2O 266.4 μl, BSA 24μl, Goat IgG 6 μl, Biotinylated Ab 3.6 μl

Wash on fluidics station using the appropriate antibody amplificationprotocol

Example 2 Characterization of Gene Expression Profiles During PlantDevelopment Using the GENECHIP®

The Arabidopsis GENECHIP® provides a method to simultaneously scan over30% of the genome for the expression profile of each gene on chip. Byusing RNA extracted from different tissue and developmental stages ofdevelopment, a scan of the entire Arabidopsis plant is achieved. Theadvantages of a gene chip in such an analysis include a global geneexpression analysis, quantitative results, a highly reproducible system,and a higher sensitivity than Northern blot analyses. Moreover, a genechip with Arabidopsis DNA has a further advantage in that theArabidopsis genome is well characterized.

Using the recently designed Arabidopsis high density oligonucleotideprobe array, a total of 8,100 Arabidopsis thaliana genes were surveyedfor temporal and developmental expression profiling. The objective wasto identify known and novel genes that are expressed in specific organs(spatial expression) or developmental stages (temporal expression versusconstitutive expression). The represented genes included approximately1,000 known full length cDNAs, a collection of approximately 500 ESTs orfull length sequences, 3,500 annotated GENBANK® genomic sequences (thetranscripts of which were confirmed by the presence of ESTs in thedatabase) and about 3,700 annotated GENBANK® sequences with a predictedtranslated open reading frame with 2 or more “hits” with a protein inthe protein database having a defined function.

Total RNA was isolated from 9 samples at different developmental stagesfor to prepare cRNA microanalysis. These samples were analyzed in 9separate GENECHIP® (see, e.g., U.S. Pat. Nos. 5,445,934, 5,744,305,5,700,305, 5,700,637, 5,945,334 and EP 619321 and EP 373203) experimentsthat included RNA from: 1) germinating seed, day 4; 2) root 2 week; 3)root adult: 4) leaf; 5) leaf adult; 6) leaf senescence; 7) stem; 8)immature siliques; and 9) flowers prior to pollen shed. The samples werehybridized to the Arabidopsis arrays and analyzed by laser scanning forrelative expression level, fold difference, organ and developmentalexpression. All genes were expressed in at least one of the samples.

Seeds of wild-type plants of Arabidopsis thaliana, ecotype Columbia,were sterilized and germinated in soil. Plants were grown in convirongrowth chambers with 12 hours of light at 22° C. 12:12 light dark cyclein metromix. Samples from leaves of 2-week, 5-week, 6-week, 8-week, and11-week old plants, and inflorescences, flowers and siliques of the6-week and 8-week old plants were collected (Table 2). In addition,4-day old seedlings and roots from 2-week, 4-week, and 5-week old plantscultured in MS liquid medium were collected. Samples collected from over30 plants were pooled and homogenized in liquid nitrogen. Total RNA wasextracted using Qiagen Rneasy column (Qiagen, Chatsworth, Calif.).

TABLE 2 germinating seedling 4 days of development germinating seedling4 days of development leaf 2 weeks after planting leaf 2 weeks afterplanting leaf 5 weeks after planting leaf 6 weeks after planting leaf 8weeks after planting leaf 11 weeks after planting root 2 weeks afterplanting root 2 weeks after planting root 5 weeks after planting root 6weeks after planting flower 5 weeks after planting flower 6 weeks afterplanting siliques 5 weeks after planting siliques 6 weeks after plantingsiliques 8-11 weeks after planting inflorescence 6 weeks after plantinginflorescence 5 weeks after planting

Total RNA (5 μg) from each sample was reverse transcribed using an oligodT₍₂₄₎ primer containing a 5′ T7 RNA polymerase promoter sequence(5′-GGCCAGTGAATT GTAATACGACTCACTATAGGGAGGCGG-(dT)₂₄-3′; SEQ ID NO:27)and SuperScript II reverse transcriptase (Life Technologies). Secondstrand of cDNA was synthesized using DNA polymerase I and DNA ligase.Biotinylated complementary RNAs (cRNAs) were in vitro transcribed by T7RNA Polymerase (ENZO BioArray High Yield RNA Transcript Labeling Kit,Enzo). cRNAs were purified using an affinity resin (Qiagen Rneasy SpinColumns) and randomly fragmented by incubating at 94° C. for 35 minutesin a buffer containing 40 mM Tris-acetate, pH 8.1, 100 mM potassiumacetate, and 30 mM magnesium acetate to produce molecules ofapproximately 35 to 200 bases.

The labeled samples were denatured at 99° C. for 5 minutes, equilibratedat 45° C. for 5 minutes, and hybridized to the Arabidopsis GENECHIP®genome array (Affymetrix) at 45° C. for 16 hours on a rotisserie at 60rpm. The hybridized arrays were then rinsed with 1.times.STT and stainedwith streptavidin phycoerythrin at 25° C. for 10 minutes twice with arinse in between. After staining, arrays were washed with 1×STT at 25°C. for 20 minutes and stained with biotinylated anti-streptavidinantibody at 25° C. for 10 minutes. The probe array was stained with SAPEat 25° C. for 10 minutes and washed with wash buffer A at 30° C. for 30minutes. All of the wash and stain procedures were completed using afluidic station (Affymetrix). The probe array was scanned twice and theintensities were averaged with a Hewlett-Packard GeneArray Scanner.

Genechip Suite 3.2 (Affymetrix) was used for data normalization. Theoverall intensity of all probe sets of each chip was scaled to 100 sothat the hybridization intensity of all arrays was equivalent. Falsepositives are defined based on experiments in which samples are split,hybridized to GENECHIP® expression arrays and the results compared. Afalse positive is indicated if a probe set is scored qualitatively as an“Increase” or “Decrease” and quantitatively as changing by at least2-fold and the average difference is greater than 25. A significantchange is defined as 2-fold change or above with an expression baselineof 25, which is determined as the threshold level according to thescaling. For example, the data from each chip was loaded into GeneSpringsoftware and analyzed for fold differences with the leaf samples. The2-week leaf samples were used to find genes expressed 4-fold or higherin the leaf sample at 2 weeks of age versus all the other tissues. Theremaining leaf samples at 5, 6, 8, and 11 weeks were not analyzed atthis stage, but were analyzed independently. The leaf sample at 5 weekswas also analyzed against all other tissues except the remaining leafsamples for genes expressed 4-fold or higher in leaf tissue at 5 weeks.The other leaf samples were analyzed in a similar fashion. This allowedthe selection of genes that were at least 4-fold elevated in expressionin a leaf sample in at least one stage of development. When these geneswere combined, there were 92 genes that were preferentially expressed inleaf tissue.

Image Analysis and Data Mining

Two text files are included in the analysis:

-   -   a. One with Absolute analysis: giving the status of each gene,        either absent or present in the samples    -   b. The other with Comparison analysis: comparing gene expression        levels between two samples

Arabidopsis Genome Array

A high-density Arabidopsis oligonucleotide array was used that includesprobes for 8,100 Arabidopsis genes and 40 probes for spiking andnegative controls. For each gene, there are 16 probe pairs (probe sets)including perfect match probes and mismatch probes for non-specificbinding control. The Arabidopsis genes are represented by known genes,predicted genes and approximately 100 clusters of ESTs. Predicted genesequences were extracted and confirmed computationally by matching thegenome sequence with ESTs and protein sequences.

The reproducibility of the array was characterized by calculation of therate of false changes (number of genes significantly changed over thetotal number of genes on the array; Lipshultz, 1999). Two cDNA andsubsequently cRNA (the antisense RNA synthesized by in vitrotranscription using cDNAs as templates in the presence of biotinylatedribonucleotides) samples were prepared in parallel from the same totalRNA samples, and hybridized to two different arrays manufactured in thesame lot or different lots. Genes that showed changes of ≧2-fold and asignal threshold above the background (calculated according to thesetting of the global scaling factor) were counted as false changes.Data from 15 pairs of array experiments indicated that false changesbetween two experiments using arrays of the same lot is 0.17% (based on8 pairs), while the false change using arrays of two different lots is0.22% (based on 7 pairs). Further analyses of these genes indicate thatthe fold change and expression levels are low and close to the threshold(Zhu and Wang, 2000).

Selected housekeeping genes are used to ensure the quality of the arrayexperiments, because the quality of the total RNA and subsequentlysynthesized cDNA and cRNA samples has direct impact on the arrayresults. Sample quality, specifically, labeled cRNA quality wasmonitored by comparing the ratio of the hybridization signal of 3′ and5′ probe sets for GAPDH and ubiqutin11. Only data with a consistent3′/5′ ratio (Zhu and Wang, 2000) was archived in the database and used.

Specific Selection Criteria

The following criteria selection were employed to identify Arabidopsisgenes that were constitutively expressed.

-   -   Baseline (background)=relative expression level of 50    -   Candidates were first selected for relative expression of ≧250        in all tissues for a given gene.    -   Relative expression range of the 346 genes which were expressed        in all tissue=250-6,765.    -   Candidate genes were selected for +/−5 fold difference in        expression=331 genes    -   Candidate genes were selected for +/−3 fold difference=276 genes    -   For 174 selected genes which met the above criteria    -   The expression for each gene was averaged:        -   ‘low’ expression=250-750; 97 genes (55.7%)        -   ‘mode rate’ expression=750-2250; 70 genes (40.2%)        -   ‘high’ expression=2250-6750; 8 genes (4.6%)    -   47 genes were selected for further analysis        -   ‘low’ expression=250-750; 21 genes (44.6%)        -   ‘mode rate’ expression=750-2250; 24 genes (51.0%)        -   ‘high’ expression=2250-6750; 3 genes (6.4%)

The following criteria were used to identify Arabidopsis genes expressedprimarily in root tissue.

-   -   Baseline (background)=relative expression level of 50    -   Candidates were first selected for relative expression of ≧300        in all tissues for a given gene excluding the germinating seed        data.    -   Candidate genes were sorted by fold difference. Root +/−3 other        tissue <10 (10 fold lower expression)    -   When the germinating seed data included was included with the 64        selected genes 39 were identified with relative expression ≧150.    -   Thirteen were selected for further analysis.

Results

Abundance Distribution of Transcripts

Knowledge of the levels of all detectable mRNA species in Arabidopsis isuseful for evaluating the complexity of the transcriptome and itscontrol. The abundance of the transcript species and their expressionlevel in 5-week-old Arabidopsis was analyzed by examining the mRNAtranscripts present in four major organs, leaves, roots, inflorescencestems, and flowers. Among 8,300 genes analyzed, over 5,000 transcriptspecies were detected in each organ. Comparison of the transcriptspresented in these organs revealed the number and percentage of thecommonly expressed and specifically expressed transcripts in each organat this stage (Table 3).

TABLE 3 Root Inflorescence Stem Leaf Flower Root 6,052 4,928 4,915 5,243Inflorescence Stem 5,399 4,828 5,036 Leaf 5,416 4,995 Flower 6,097Specific 426 55 89 380

Expression measurements (average signal difference between perfect-matchprobes and mismatch probes) of the genes in each organ were examined.Data were collected and log transformed, then plotted against theirfrequencies. A normal distribution of the transcript abundance wasrevealed for all four organs. The median of the distributions is similarto the profiles of yeast, mammalian, and E. coli (Lockhart and Winzler,2000). Overall, the transcription profile is more complex in flowersthan in the vegetative organs. It is evidenced by the elevatedfrequencies in almost every level of transcription. Root has the mostcomplex profile among the vegetative organs, while leaf andinflorescence stem have very similar and simpler profiles.

Constitutive and Organ Differential Gene Expression

The composition of the constitutively and organ differentially expressedtranscripts were characterized. A total of 347 constitutive expressedgenes with median or high-level transcripts were selected from thecommonly expressed gene pool (Table 4). These genes are constantlyexpressed above median expression level (average difference greater than500) for all organs and developmental stages examined. Functionalcategorization indicated that majority of the known constitutive genesare involved in metabolism (28%) and ribosomal protein synthesis (15%),followed by genes involving transcription (8%), signaling (6%),transport (5%), membrane (5%), synthases (5%), membrane (5%), and stressand defense related (7%). About 15% of the genes identified have nofunction assigned.

TABLE 4 Constitutively expressed Arabidopsis sequences and theircorresponding genes. Gene ID Accession # on chip Affy # DescriptionA45785.1_S_AT A45785.1 19852_s_at emb|CAA02840.1|(A45785) unnamedprotein product [Arabidopsis thaliana] AB003522.2_AT AB003522.2 12381_atdbj|BAA84392.1|(AP000423) ATPase beta subunit [Arabidopsis thaliana]AB004872.6_S_AT AB004872.6 15997_s_at dbj|BAA23547.1|(AB004872) COR47[Arabidopsis thaliana] AB005560_S_AT AB004872.6 15630_s_atdbj|BAA22504.1|(AB005560) AtGDI2 [Arabidopsis thaliana] AB006693.1_ATAB006693.1 17438_at dbj|BAA24536.1|(AB006693) spermidine synthase[Arabidopsis thaliana] AB008105_S_AT AB008105 17044_s_atdbj|BAA32420.1|(AB008105) ethylene responsive element binding factor 3[Arabidopsis thaliana] AB008487_S_AT AB008487 15127_s_atdbj|BAA31143.1|(AB010915) responce regulator1 [Arabidopsis thaliana]AB008854_S_AT AB008854 14719_s_at dbj|BAA25248.1|(AB008854) 3-ketoacyl-CoA thiolase [Arabidopsis thaliana] AB010946_S_AT AB01094615200_s_at dbj|BAA24804.1|(AB010946) AtRer1B [Arabidopsis thaliana]AB011545_S_AT AB011545 15163_s_at dbj|BAA32735.1|(AB011545) GF14 mu[Arabidopsis thaliana] thaliana] AB017643_S_AT AB017643 15164_s_atgb|AAC14411.1|(AF049236) putative acyl-coA dehydrogenase [Arabidopsisthaliana] AB021858_S_AT AB021858 16540_s_at dbj|BAA77759.1|(AB021858)plastid heme oxygenase [Arabidopsis thaliana] AB024282_S_AT AB02428215128_s_at emb|CAB71074.1|(AL132962) cysteine synthase AtcysC1[Arabidopsis thaliana] AB027151.2_S_AT AB027151.2 19179_s_atemb|CAB43659.1|(AL050352) threonine synthase [Arabidopsis thaliana]AC000103.25_S_AT AC000103.25 20709_s_at gb|AAB61517.1|(AC000103)F21J9.25 [Arabidopsis thaliana] AC000104.10_R_AT AC000104.10 13076_r_atgb|AAB70426.1|(AC000104) Strong similarity to 60S ribosomal protein L17(gb|X01694). EST gb|AA042332 comes from this gene. [Arabidopsisthaliana] AC000104.26_AT AC000104.26 12771_at gb|AAB70434.1|(AC000104)F19P19.13 [Arabidopsis thaliana] AC000106.13_S_AT AC000106.13 17900_s_atgb|AAB70401.1|(AC000106) Similar to Glycine SRC2 (gb|AB000130). ESTsgb|H76869, gb|T21700, gb|ATTS5089 come from this gene. [Arabidopsisthaliana] AC000132.16_S_AT AC000132.16 16531_s_atgb|AAC33220.1|(AC003970) Putative ribosomal protein L21 [Arabidopsisthaliana] gb|AA395597, gb|ATTS5197 come from this gene. [Arabidopsisthaliana] AC000132.6_AT AC000132.6 16420_at gb|AAB60721.1|(AC000132)Similar to elongation factor 1-gamma (gb|EF1G_XENLA). ESTs gb|T20564,gb|T45940, gb|T04527 come from this gene. [Arabidopsis thaliana]AC002131.48_S_AT AC002131.48 12750_s_at gb|AAC17620.1|(AC002131)Identical to aspartic proteinase cDNA gb|U51036 from A. thaliana. ESTsgb|N96313, gb|T21893, gb|R30158, gb|T21482, gb|T43650, gb|R64749,gb|R65157, gb|T88269, gb|T44552, gb|T22542, gb|T76533, gb|T44350,gb|Z34591, gb|AA728734, g AC002329.46_AT AC002329.46 13074_atemb|CAA54095.1|(X76651) ribosomal protein S4 [Solanum tuberosum]AC002330.39_AT AC002330.39 13574_at gb|AAC78269.1|AAC78269 (AC002330)putative vacuolar ATPase [Arabidopsis thaliana] AC002332.100_ATAC002332.100 13105_at gb|AAB80655.1|(AC002332) 60S ribosomal protein L23[Arabidopsis thaliana] AC002332.71_AT AC002332.71 17435_atgb|AAB80652.1|(AC002332) putative PRP19-like spliceosomal protein[Arabidopsis thaliana] AC002334.110_G_AT AC002334.110 16940_g_atgb|AAC04922.1|(AC002334) putative synaptobrevin [Arabidopsis thaliana]AC002336.101_G_AT AC002336.101 12809_g_at gb|AAB87594.1|(AC002336) 40Sribosomal protein S26 [Arabidopsis thaliana] AC002339.51_AT AC002339.5116507_at gb|AAC02764.1|(AC002339) 40S ribosomal protein S2 [Arabidopsisthaliana] AC002343.3_AT AC002343.3 16447_at gb|AAB63606.1|(AC002343)HSP90 isolog [Arabidopsis thaliana] AC002521.146_AT AC002521.14616917_at gb|AAC05346.1|(AC002521) putative ubiquitin-conjugating enzymeE2 [Arabidopsis AC002561.51_AT AC002561.51 18655_atgb|AAB88646.1|(AC002561) unknown protein [Arabidopsis thaliana]AC003672.64_S_AT AC003672.64 20425_s_at gb|AAC27463.1|(AC003672)putative small GTP-binding protein [Arabidopsis thaliana]AC003981.34_S_AT AC003981.34 16523_s_at gb|AAC14060.1|(AC003981)F22O13.34 [Arabidopsis thaliana] AC004077.166_S_AT AC004077.16617004_s_at gb|AAC26708.1|(AC004077) 60S ribosomal protein L18A[Arabidopsis thaliana] AC004165.105_AT AC004165.105 13125_atgb|AAC16961.1|(AC004165) putative ubiquitin activating enzyme (UBA1)[Arabidopsis AC004218.83_S_AT AC004218.83 13616_s_atgb|AAC27837.1|(AC004218) 60S ribosomal protein L23A [Arabidopsisthaliana] AC004393.22_AT AC004393.22 16953_at gb|AAC18792.1|(AC004393)Similar to ribosomal protein L17 gb|X62724 from Hordeum vulgare. ESTsgb|Z34728, gb|F19974, gb|T75677 and gb|Z33937 come from this gene.[Arabidopsis thaliana] AC004401.119_AT AC004401.119 13594_atgb|AAC17825.1|(AC004401) unknown protein [Arabidopsis thaliana]AC004401.140_AT AC004401.140 12767_at gb|AAB87096.2|(AC002391) unknownprotein [Arabidopsis thaliana] AC004450.11_AT AC004450.11 18882_atgb|AAC64298.1|(AC004450) 3- isopropylmalate dehydratase, small subunit[Arabidopsis thaliana] AC004450.83_AT AC004450.83 18262_atgb|AAC64306.1|(AC004450) unknown protein [Arabidopsis thaliana]AC004481.84_AT AC004481.84 13102_at gb|AAC27401.1|(AC004481) putativeprotein transport protein SEC61 alpha subunit [Arabidopsis thaliana]AC004557.10_AT AC004557.10 17436_at gb|AAC80610.1|(AC004557) F17L21.10[Arabidopsis thaliana] AC004557.20_AT AC004557.20 17374_atgb|AAC80620.1|(AC004557) F17L21.20 [Arabidopsis thaliana] AC004557.8_ATAC004557.8 18874_at gb|AAC80608.1|(AC004557) F17L21.8 [Arabidopsisthaliana] AC004665.121_S_AT AC004665.121 18629_s_atgb|AAC28542.1|(AC004665) remorin [Arabidopsis thaliana] AC004665.31_S_ATAC004665.31 15977_s_at gb|AAC28529.1|(AC004665) aquaporin (plasmamembrane intrinsic protein 1B) [Arabidopsis thaliana] AC004669.34_ATAC004669.34 16430_at gb|AAC20720.1|(AC004669) glutathione S-transferase[Arabidopsis thaliana] AC004747.160_S_AT AC004747.160 15506_s_atgb|AAC31239.1|(AC004747) unknown protein [Arabidopsis thaliana]AC005169.214_AT AC005169.214 18221_at gb|AAC62141.1|(AC005169) 40Sribosomal protein S30 [Arabidopsis thaliana] AC005169.221_ATAC005169.221 18283_at gb|AAC62149.1|(AC005169) putative ribosomalprotein L28 [Arabidopsis thaliana] AC005287.20_S_AT AC005287.2016027_s_at gb|AAD25605.1|AC005287_7 (AC005287) Eukaryotic InitiationFactor 4A-2 [Arabidopsis thaliana] AC005287.52_AT AC005287.52 14073_atNo hits found less than or equal to 1e−15. AC005309.201_I_ATAC005309.201 15570_i_at gb|AAC63650.1|(AC005309) unknown protein[Arabidopsis thaliana] AC005309.64_S_AT AC005309.64 16009_s_atgb|AAC63629.1|(AC005309) glutathione S-transferase (GST6) [Arabidopsisthaliana] AC005388.6_S_AT AC005388.6 12783_s_at gb|AAC64875.1|(AC005388)Identical to gb|L14814 DNA for tissue-specific acyl carrier proteinisoform 2 from A. thaliana. ESTs gb|AA597351, gb|T41805, gb|H36871,gb|R30210, gb|AA042549, gb|Z47650, gb|H76304 and gb|AA597348 come fromthis gene. [Arabidops AC005397.40_S_AT AC005397.40 16471_s_atgb|AAC62877.1|(AC005397) eukaryotic translation initiation factor 3delta subunit [Arabidopsis thaliana] AC005662.30_S_AT AC005662.3016952_s_at gb|AAC78532.1|(AC005662) calmodulin-like protein [Arabidopsisthaliana] AC005679.10_S_AT AC005679.10 12775_s_atgb|AAC83021.1|(AC005679) Identical to gb|U65638 Arabidopsis thalianavacuolar type ATPase subunit A mRNA. ESTs gb|N96435, gb|N96106,gb|N96189, gb|N96091, gb|AA042286, gb|F14324, gb|W43643, gb|N96027,gb|N96299, gb|R29943, gb|T43460, gb|T43544, gb|T2247 AC005727.191_ATAC005727.191 16901_at gb|AAC79595.1|(AC005727) unknown protein[Arabidopsis thaliana] AC005824.107_AT AC005824.107 16527_atgb|AAC73028.1|(AC005824) 60S acidic ribosomal protein P2 [Arabidopsisthaliana] AC005824.114_AT AC005824.114 17910_at gb|AAC73029.1|(AC005824)60S acidic ribosomal protein P2 [Arabidopsis thaliana] AC005824.21_ATAC005824.21 13089_at gb|AAC73015.1|(AC005824) putative dTDP-glucose 4-6-dehydratase [Arabidopsis thaliana] AC005896.150_S_AT AC005896.15018603_s_at gb|AAC98060.1|(AC005896) putative protein translocase[Arabidopsis thaliana] AC005897.156_S_AT AC005897.156 13572_s_atgb|AAC97246.1|(AC005897) 10- formyltetrahydrofolate synthetase[Arabidopsis thaliana] AC005936.95_AT AC005936.95 16416_atgb|AAC97221.1|(AC005936) protease inhibitor II [Arabidopsis thaliana]AC005990.10_AT AC005990.10 13069_at gb|AAC98042.1|(AC005990) Strongsimilarity to gb|M95166 ADP- ribosylation factor from Arabidopsisthaliana. ESTs gb|Z25826, gb|R90191, gb|N65697, gb|AA713150, gb|T46332,gb|AA040967, gb|AA712956, gb|T46403, gb|T46050, gb|AI100391 andgb|Z25043 come from AC006068.93_AT AC006068.93 18645_atgb|AAD15447.1|(AC006068) unknown protein [Arabidopsis thaliana]AC006085.15_AT AC006085.15 20562_at gb|AAD30634.1|AC006085_7 (AC006085)Unknown protein [Arabidopsis thaliana] AC006200.119_AT AC006200.11913132_at gb|AAD14525.1|(AC006200) 60S ribosomal protein L7 [Arabidopsisthaliana] AC006201.107_S_AT AC006201.107 16924_s_atgb|AAD20124.1|(AC006201) 60S ribosomal protein L2 [Arabidopsis thaliana]AC006223.65_AT AC006223.65 14089_at gb|AAD15390.1|(AC006223) putativehydrolase [Arabidopsis thaliana] AC006234.156_AT AC006234.156 14099_atgb|AAD20913.1|(AC006234) unknown protein [Arabidopsis thaliana]AC006260.52_AT AC006260.52 12769_at gb|AAD18142.1|(AC006260) aquaporin(plasma membrane intrinsic protein 2B) [Arabidopsis thaliana]AC006264.30_AT AC006264.30 13095_at gb|AAD29800.1|AC006264_8 (AC006264)putative signal sequence receptor, alpha subunit AC006300.112_ATAC006300.112 16948_at gb|AAD20708.1|(AC006300) putative glucoseregulated repressor protein [Arabidopsis thaliana] AC006300.70_ATAC006300.70 16487_at gb|AAD20704.1|(AC006300) putative dioxygenase[Arabidopsis thaliana] AC006403.110_AT AC006403.110 18223_atgb|AAD18124.1|(AC006403) unknown protein [Arabidopsis thaliana]AC006438.21_AT AC006438.21 12749_at gb|AAD41971.1|AC006438_3 (AC006438)similar to cold acclimation protein WCOR413 [Triticum aestivum][Arabidopsis thaliana] AC006526.57_AT AC006526.57 14103_at No hits foundless than or equal to 1e−15. AC006532.47_AT AC006532.47 19940_atgb|AAD20090.1|(AC006532) putative endosomal protein [Arabidopsisthaliana] AC006577.32_AT AC006577.32 16941_at gb|AAD25780.1|AC006577_16(AC006577) Similar to gb|U55861 RNA binding protein nucleolysin (TIAR)from Mus musculus and contains several PF|00076 RNA recognition motifdomains. ESTs gb|T21032 and gb|T44127 come from this gene. [Arabidopsisthaliana] AC006585.146_AT AC006585.146 14565_atgb|AAD23019.1|AC006585_14 (AC006585) putative steroid binding protein[Arabidopsis thaliana] AC006586.141_AT AC006586.141 17390_atgb|AAD22696.1|AC006586_5 (AC006586) 40S ribosomal protein S16[Arabidopsis thaliana] AC006592.150_S_AT AC006592.150 15980_s_atemb|CAA47427.1|(X67034) Athb-6 [Arabidopsis thaliana] AC006841.122_ATAC006841.122 19650_at gb|AAD23699.1|AC006841_15 (AC006841) coatomeralpha subunit [Arabidopsis thaliana] AC006919.140_AT AC006919.14012742_at gb|AAD24635.1|AC006919_15 (AC006919) enolase (2-phospho-D-glycerate hydroylase) [Arabidopsis AC006919.171_AT AC006919.171 13070_atgb|AAD24640.1|AC006919_20 (AC006919) putative pyruvate kinase[Arabidopsis thaliana] AC006921.52_AT AC006921.52 16511_atgb|AAD21434.1|(AC006921) unknown protein [Arabidopsis thaliana]AC006922.106_AT AC006922.106 12412_at gb|AAD31573.1|AC006922_5(AC006922) putative s- adenosylmethionine synthetase [Arabidopsisthaliana] AC006922.28_S_AT AC006922.28 15962_s_atgb|AAD31569.1|AC006922_1 (AC006922) putative aquaporin (tonoplastintrinsic protein gamma) AC006929.77_AT AC006929.77 13150_atgb|AAD21502.1|(AC006929) putative rubisco subunit binding- protein alphasubunit [Arabidopsis thaliana] AC006951.208_S_AT AC006951.208 13107_s_atgb|AAD25839.1|AC006951_18 (AC006951) 40S ribosomal protein S17[Arabidopsis thaliana] AC007017.278_S_AT AC007017.278 20024_s_atgb|AAD21476.1|(AC007017) unknown protein [Arabidopsis thaliana]AC007019.105_AT AC007019.105 16022_at gb|AAD20405.1|(AC007019) putativeATP synthase [Arabidopsis thaliana] AC007070.167_AT AC007070.16713166_at emb|CAA64728.1|(X95458) ribosomal protein L39 [Zea mays]AC007071.72_AT AC007071.72 16933_at gb|AAD24852.1|AC007071_24 (AC007071)40S ribosomal protein; contains C-terminal domain [Arabidopsis thaliana]AC007119.88_AT AC007119.88 13080_at gb|AAD23647.1|AC007119_13 (AC007119)40S ribosomal protein S25 [Arabidopsis thaliana] AC007135.50_ATAC007135.50 16919_at gb|AAD26971.1|AC007135_8 (AC007135) 40S ribosomalprotein S14 [Arabidopsis thaliana] AC007138.25_S_AT AC007138.2512797_s_at gb|AAD22647.1|AC007138_11 (AC007138) S-adenosylmethioninesynthase 2 [Arabidopsis thaliana] AC007170.48_AT AC007170.48 17857_atgb|AAD25640.1|AC007170_2 (AC007170) cytoplasmic aconitate hydratase[Arabidopsis thaliana] AC007195.93_I_AT AC007195.93 16969_i_atgb|AAA99933.1|(L44581) vacuolar H+-pumping ATPase 16 kDa proteolipid[Arabidopsis [Arabidopsis thaliana] AC007357.17_S_AT AC007357.1713104_s_at emb|CAA74029.1|(Y13695) multicatalytic endopeptidase complex,proteasome precursor, beta subunit [Arabidopsis thaliana] AC007576.5_ATAC007576.5 12781_at gb|AAD39279.1|AC007576_2 (AC007576) Unknown protein[Arabidopsis thaliana] AC007659.93_R_AT AC007659.93 13169_r_atgb|AAD32831.1|AC007659_13 (AC007659) putative GATA-type zinc fingertranscription factor [Arabidopsis thaliana] AF000657.40_AT AF000657.4019623_at gb|AAB72175.1|(AF000657) cytochrome C [Arabidopsis thaliana]AF001394_S_AT AF001394 15600_s_at gb|AAD00895.1|(AF001394) fatty aciddesaturase/cytochrome b5 fusion protein [Arabidopsis thaliana]AF003096_F_AT AF003096 14723_f_at gb|AAC49769.1|(AF003096) AP2 domaincontaining protein RAP2.3 [Arabidopsis thaliana] AF003105.1_ATAF003105.1 17858_at gb|AAC49778.1|(AF003105) AP2 domain containingprotein RAP2.12 [Arabidopsis thaliana] AF004216_S_AT AF004216 15205_s_atgb|AAC49749.1|(AF004216) ethylene-insensitive3 [Arabidopsis thaliana]AF004393_S_AT AF004393 14714_s_at gb|AAB62692.1|(AF004393) salt- stressinduced tonoplast intrinsic protein [Arabidopsis thaliana]AF013294.25_S_AT AF013294.25 18650_s_at gb|AAB62867.1|(AF013294) AT0ZI1gene product [Arabidopsis thaliana] AF013294.35_AT AF013294.35 18573_atgb|AAB62855.1|(AF013294) similar to acidic ribosomal protein p1[Arabidopsis thaliana] AF013959.4_AT AF013959.4 16436_atgb|AAB67234.1|(AF013959) metallothionein-like protein [Arabidopsisthaliana] AF017641_S_AT AF017641 15165_s_at gb|AAC17844.1|(AF017641)nucleoside diphosphate kinase type 1 [Arabidopsis AF017991_S_AT AF01799115150_s_at gb|AAB97312.1|(AF017991) salt stress inducible small GTPbinding protein Ran1 AF027172.3_S_AT AF027172.3 16906_s_atgb|AAC39334.1|(AF027172) cellulose synthase catalytic subunit[Arabidopsis thaliana] AF027174_S_AT AF027174 15603_s_atgb|AAC39336.1|(AF027174) cellulose synthase catalytic subunit[Arabidopsis thaliana] AF034387_S_AT AF034387 14727_s_atgb|AAC33264.1|(AF034387) AFT protein [Arabidopsis thaliana]AF034694_S_AT AF034694 16544_s_at gb|AAB87692.1|(AF034694) ribosomalprotein L23a [Arabidopsis thaliana] AF043519_S_AT AF043519 15130_s_atgb|AAC95161.1|(AC005970) 20S proteasome subunit (PAA2) [Arabidopsisthaliana] AF043528_S_AT AF043528 16546_s_at gb|AAC32064.1|(AF043528) 20Sproteasome subunit PAG1 [Arabidopsis thaliana] AF044265_S_AT AF04426515668_s_at gb|AAC00512.1|(AF044265) nucleoside diphosphate kinase 3[Arabidopsis thaliana] AF044313_S_AT AF044313 14717_s_atgb|AAC05742.1|(AF044313) anion channel protein [Arabidopsis thaliana]AF059294_S_AT AF059294 14736_s_at gb|AAF26761.1|AC007396_10 (AC007396)T4O12.15 [Arabidopsis thaliana] protein in budding yeast [Arabidopsisthaliana] AF061519_S_AT AF061519 15581_s_at gb|AAD10208.1|(AF061519)copper/zinc superoxide dismutase [Arabidopsis thaliana] AF062485.1_ATAF062485.1 17468_at gb|AAC29067.1|(AF062485) cellulose synthase[Arabidopsis thaliana] AF063901_S_AT AF063901 14737_s_atgb|AAC26854.1|(AF063901) alanine: glyoxylate aminotransferase;transaminase [Arabidopsis thaliana] AF069299.19_AT AF069299.19 16925_atgb|AAC19305.1|(AF069299) similar to ribosomal protein S13 (Pfam;S15.hmm, score: 78.35); identical to Arabidopsis 40S ribosomal proteinS13 (fragment) (SW: P49203A) except the first 32 amino acids aredifferent [Arabidopsis thaliana] AF074375_S_AT AF074375 15114_s_atgb|AAC83240.1|(AF073875) endo- 1,4-beta-D-glucanase KORRIGAN[Arabidopsis thaliana] AF076484_S_AT AF076484 16627_s_atgb|AAD04627.1|(AF108660) CYT1 protein [Arabidopsis thaliana]AF076641.2_AT AF076641.2 16977_at gb|AAD46064.1|AF076641_1 (AF076641)homeodomain leucine- zipper protein ATHB16 [Arabidopsis thaliana]AF077528_S_AT AF077528 15152_s_at gb|AAB72116.1|(U69533) AtKAP alpha[Arabidopsis thaliana] AF080120.11_S_AT AF080120.11 16935_s_atgb|AAC35545.1|(AF080120) similar to vacuolar ATPases [Arabidopsisthaliana] thaliana] AF082565_S_AT AF082565 15639_s_atgb|AAD29109.1|AF082565_1 (AF082565) ATP dependent copper transporter[Arabidopsis thaliana] AF083336.2_S_AT AF083336.2 16932_s_atgb|AAD10030.1|(AF083337) ribosomal protein S27 [Arabidopsis thaliana]AF083337.3_S_AT AF083337.3 16931_s_at gb|AAD10030.1|(AF083337) ribosomalprotein S27 [Arabidopsis thaliana] AF118822_F_AT AF118822 16080_f_atgb|AAD20612.1|(AF118822) senescence-associated protein [Arabidopsisthaliana] AF123253.3_I_AT AF123253.3 20459_i_atemb|CAB43915.1|(AL078470) AIM1 protein [Arabidopsis thaliana]AF136152_S_AT AF136152 15643_s_at gb|AAD39465.1|AF136152_1 (AF136152)PUR alpha-1 [Arabidopsis thaliana] AF144387_AT AF144387 12857_atgb|AAD35005.1|AF144387_1 (AF144387) thioredoxin-like 1 [Arabidopsisthaliana] AF167983_S_AT AF167983 15210_s_at gb|AAC26685.1|(AC004077)putative pyruvate dehydrogenase E1 beta subunit [Arabidopsis thaliana]AF181688_R_AT AF181688 17994_r_at gb|AAF24609.1|AC010870_2 (AC010870)vacuolar membrane ATPase subunit G (AVMA10) [Arabidopsis thaliana]AF181966_AT AF181966 17996_at gb|AAD55787.1|AF181966_1 (AF181966)methylenetetrahydrofolate reductase MTHFR1 [Arabidopsis thaliana]AF186847_S_AT AF186847 18000_s_at gb|AAF03749.1|AF186847_1 (AF186847)TIM17 [Arabidopsis thaliana] AGO1_S_AT AGO1 12877_s_atgb|AAD49755.1|AC007932_3 (AC007932) Identical to gb|U91995 Argonauteprotein from Arabidopsis AJ001342.2_S_AT AJ001342.2 16923_s_atemb|CAA18846.1|(AL023094) Putative S-phase-specific ribosomal protein[Arabidopsis thaliana] AJ001397_S_AT AJ001397 18011_s_atdbj|BAA22504.1|(AB005560) AtGDI2 [Arabidopsis thaliana] AJ006787.1_ATAJ006787.1 19224_at emb|CAA07251.1|(AJ006787) putative phytochelatinsynthetase [Arabidopsis thaliana] AJ010456.2_AT AJ010456.2 17470_atemb|CAA09195.1|(AJ010456) RNA helicase [Arabidopsis thaliana]AJ010505_S_AT AJ010505 18018_s_at emb|CAB54830.1|(AJ010505) cysteinesynthase [Arabidopsis thaliana] AJ011628_I_AT AJ011628 18032_i_atemb|CAB56580.1|(AJ011628) squamosa promoter binding protein- like 1[Arabidopsis thaliana] AJ012571.2_S_AT AJ012571.2 16012_s_atemb|CAA10060.1|(AJ012571) glutathione transferase [Arabidopsis thaliana]AJ131205_AT AJ131205 18047_at emb|CAA10320.1|(AJ131205) mitochondrialNAD-dependent malate dehydrogenase [Arabidopsis thaliana]AL021636.178_AT AL021636.178 16499_at emb|CAA16587.1|(AL021636) putativeprotein [Arabidopsis thaliana] AL021687.199_AT AL021687.199 19677_atemb|CAA16709.1|(AL021687) putative protein [Arabidopsis thaliana]AL021712.156_AT AL021712.156 20559_at emb|CAA16781.1|(AL021712) putativeprotein [Arabidopsis thaliana] AL021811.156_AT AL021811.156 12776_atemb|CAA16969.1|(AL021811) putative protein [Arabidopsis thaliana]AL021890.14_AT AL021890.14 13591_at emb|CAA17148.1|(AL021890) putativeprotein [Arabidopsis thaliana] AL021890.209_S_AT AL021890.209 12752_s_atemb|CAA17163.1|(AL021890) peroxidase prxr1 [Arabidopsis thaliana]AL022023.145_S_AT AL022023.145 16905_s_at emb|CAA17773.1|(AL022023)catalase [Arabidopsis thaliana] AL022141.10_S_AT AL022141.10 16976_s_atemb|CAA18507.1|(AL022373) ribosomal protein L2 [Arabidopsis thaliana]AL022224.182_S_AT AL022224.182 16021_s_at emb|CAA18251.1|(AL022224)endomembrane-associated protein [Arabidopsis thaliana] AL022224.72_ATAL022224.72 13122_at emb|CAA18240.1|(AL022224) putative protein[Arabidopsis thaliana] AL022373.153_AT AL022373.153 12802_atemb|CAA18498.1|(AL022373) DnaJ- like protein [Arabidopsis thaliana]AL022580.188_AT AL022580.188 17878_at emb|CAA18628.1|(AL022580) putativepectinacetylesterase protein [Arabidopsis thaliana] AL023094.216_S_ATAL023094.216 12234_s_at emb|CAA18841.1|(AL023094) putative ribosomalprotein S16 [Arabidopsis thaliana] AL023094.323_S_AT AL023094.32316515_s_at emb|CAA18849.1|(AL023094) putative protein [Arabidopsisthaliana] AL031326.138_AT AL031326.138 17931_atemb|CAA20461.1|(AL031326) water channel-like protein [Arabidopsisthaliana] AL034567.189_AT AL034567.189 13088_atemb|CAA22574.1|(AL034567) ubiquinol-cytochrome c reductase- like protein[Arabidopsis thaliana] AL035356.123_AT AL035356.123 13097_atemb|CAA22994.1|(AL035356) putative protein [Arabidopsis thaliana]AL035394.117_AT AL035394.117 17384_at emb|CAA23029.1|(AL035394) putativeprotein [Arabidopsis thaliana] AL035440.191_S_AT AL035440.191 13133_s_atemb|CAB36530.1|(AL035440) ubiquitin-like protein [Arabidopsis thaliana]AL035440.447_AT AL035440.447 17011_at emb|CAB36546.1|(AL035440) putativeDNA binding protein [Arabidopsis thaliana] AL035440.66_AT AL035440.6618661_at emb|CAB36517.1|(AL035440) putative protein [Arabidopsisthaliana] AL035526.101_S_AT AL035526.101 13073_s_atemb|CAB37458.1|(AL035526) ribosomal protein L11, cytosolic [Arabidopsisthaliana] AL035540.348_S_AT AL035540.348 19961_s_atgb|AAB24074.1|(S47408) glycine- rich protein, atGRP {clone atGRP-2}[Arabidopsis AL035540.94_AT AL035540.94 12804_atemb|CAB37507.1|(AL035540) probable H+-transporting ATPase [Arabidopsisthaliana] AL035656.126_AT AL035656.126 17459_atemb|CAB38614.1|(AL035656) putative protein [Arabidopsis thaliana]AL035679.13_S_AT AL035679.13 16967_s_at gb|AAA99933.1|(L44581) vacuolarH+-pumping ATPase 16 kDa proteolipid [Arabidopsis [Arabidopsis thaliana]AL035679.232_AT AL035679.232 18905_at emb|CAB38828.1|(AL035679) putativeproton pump [Arabidopsis thaliana] AL035680.110_S_AT AL035680.11017429_s_at emb|CAB38843.1|(AL035680) translation initiation factor[Arabidopsis thaliana] AL035680.53_AT AL035680.53 13578_atemb|CAB38839.1|(AL035680) ribosomal protein L14-like protein[Arabidopsis thaliana] AL035709.87_AT AL035709.87 17389_atemb|CAB38931.1|(AL035709) putative protein [Arabidopsis thaliana]AL049171.158_AT AL049171.158 20180_at No hits found less than or equalto 1e−15. AL049171.25_AT AL049171.25 17005_at emb|CAB38952.1|(AL049171)putative ribosomal protein [Arabidopsis thaliana] AL049480.178_ATAL049480.178 13940_at emb|CAB39610.1|(AL049480) putative acidicribosomal protein [Arabidopsis thaliana] AL049608.184_AT AL049608.18412813_at emb|CAB40778.1|(AL049608) putative protein [Arabidopsisthaliana] AL050300.15_F_AT AL050300.15 13129_f_atemb|CAB43405.1|(AL050300) ubiquitin/ribosomal protein CEP52 [Arabidopsisthaliana] AL050300.27_AT AL050300.27 16920_at emb|CAB43407.1|(AL050300)putative ribosomal protein S14 [Arabidopsis thaliana] AL050398.4_ATAL050398.4 19133_at emb|CAB43690.1|(AL050398) H+- transportingATPase-like protein [Arabidopsis thaliana] AL078464.37_AT AL078464.3714108_at emb|CAB43836.1|(AL078464) putative protein [Arabidopsisthaliana] AL078468.11_AT AL078468.11 18330_at emb|CAB43885.1|(AL078468)acyl- CoA synthetase-like protein [Arabidopsis thaliana]AL078637.47_S_AT AL078637.47 12803_s_at emb|CAB45057.1|(AL078637)putative protein [Arabidopsis thaliana] AL096856.7_AT AL096856.713093_at emb|CAB51061.1|(AL096856) B12D- like protein [Arabidopsisthaliana] AL096860.157_AT AL096860.157 13079_atemb|CAB51209.1|(AL096860) 40S RIBOSOMAL PROTEIN S20 homolog [Arabidopsisthaliana] AOS_S_AT AOS 12881_s_at emb|CAA63266.1|(X92510) allene oxidesynthase [Arabidopsis thaliana] AP000423_AT AP000423 12847_atdbj|BAA84366.1|(AP000423) orf within trnK intron [Arabidopsis thaliana]APX3_S_AT APX3 12885_s_at emb|CAA66640.1|(X98003) ascorbate peroxidase[Arabidopsis thaliana] ATADHIII_AT ATADHIII 12893_atemb|CAA57973.1|(X82647) class III ADH, glutathione-dependentformaldehyde dehydrogenase. [Arabidopsis thaliana] ATERF3_S_AT ATERF312906_s_at dbj|BAA32420.1|(AB008105) ethylene responsive element bindingfactor 3 [Arabidopsis thaliana] ATHADPRFA_S_AT ATHADPRFA 15677_s_atgb|AAA32729.1|(M95166) ADP- ribosylation factor [Arabidopsis thaliana]ATHAVAP_S_AT ATHAVAP 15191_s_at gb|AAA99933.1|(L44581) vacuolarH+-pumping ATPase 16 kDa proteolipid [Arabidopsis [Arabidopsis thaliana]ATHAVAPA_S_AT ATHAVAPA 15584_s_at gb|AAD26493.1|AC007195_7 (AC007195)putative vacuolar proton- ATPase 16 kDa proteolipid [Arabidopsisthaliana] ATHAVAPC_S_AT ATHAVAPC 16145_s_at gb|AAD38803.1|AF153677_1(AF153677) vacuolar H+-pumping ATPase 16 kDa subunit c isoform 4thaliana] ATHD12AAA_S_AT ATHD12AAA 15134_s_at gb|AAA32782.1|(L26296)delta-12 desaturase [Arabidopsis thaliana] ATHDYNAGTP_S_AT ATHDYNAGTP15585_s_at gb|AAB63528.1|(L36939) dynamin- like GTP binding protein[Arabidopsis thaliana] ATHERD13_S_AT ATHERD13 15193_s_atgb|AAC20721.1|(AC004669) glutathione S-transferase [Arabidopsisthaliana] ATHERD15_S_AT ATHERD15 15104_s_at gb|AAC23728.1|(AC004625)dehydration-induced protein (ERD15) [Arabidopsis thaliana]ATHGFPSIA_S_AT ATHGFPSIA 14734_s_at gb|AAA32799.1|(L09110) GF14 psichain [Arabidopsis thaliana] ATHHMG1_AT ATHHMG1 12920_atgb|AAA32814.1|(L19261) hydroxymethylglutaryl CoA reductase [Arabidopsisthaliana] ATHHMGCOAR_S_AT ATHHMGCOAR 12921_s_at emb|CAA33139.1|(X15032)hydroxy methylglutaryl CoA reductase (AA 1-592) ATHMERI5B_S_AT ATHMERI5B15614_s_at emb|CAB52471.1|(AL109796) xyloglucan endo-1,4-beta-D-glucanase precursor [Arabidopsis thaliana] ATHMTMACP_S_AT ATHMTMACP16574_s_at gb|AAB96840.1|(L23574) acyl carrier protein precursor[Arabidopsis thaliana] ATHPRPHC_S_AT ATHPRPHC 15119_s_atgb|AAD10854.1|(U60135) serine/threonine protein phosphatase 2A-3catalytic ATHRP28A_S_AT ATHRP28A 16577_s_at gb|AAA32862.1|(L09755)ribosomal protein S28 [Arabidopsis thaliana] ATHRPCA_S_AT ATHRPCA15155_s_at gb|AAA66160.1|(M32654) ribosomal protein [Arabidopsisthaliana] ATHSAR1_S_AT ATHSAR1 15617_s_at gb|AAA56991.1|(M90418)formerly called HAT24; synaptobrevin-related protein [Arabidopsisthaliana] ATORNCARB_S_AT ATORNCARB 15213_s_at emb|CAA04115.1|(AJ000476)Ornithine carbamoyltransferase [Arabidopsis thaliana] ATTHIRED2_S_ATATTHIRED2 13184_s_at gb|AAC49351.1|(U35640) thioredoxin h [Arabidopsisthaliana] ATTHIRED3_AT ATTHIRED3 13185_at emb|CAA84612.1|(Z35475)thioredoxin [Arabidopsis thaliana] ATU01955_S_AT ATU01955 15135_s_atgb|AAF27153.1|AC016529_16 (AC016529) putative 40S ribosomal protein SA(laminin receptor-like ATU09137_S_AT ATU09137 15156_s_atgb|AAA52225.1|(U09137) pyruvate dehydrogenase E1 beta subunit[Arabidopsis thaliana] ATU15108_S_AT ATU15108 17078_s_atgb|AAA50250.1|(U15108) metallothionein-like protein [Arabidopsisthaliana] ATU15130_S_AT ATU15130 15157_s_at No hits found. ATU18410_S_ATATU18410 16156_s_at gb|AAD15575.1|(AC006340) auxin- regulated protein(IAA8) [Arabidopsis thaliana] ATU18675_S_AT ATU18675 15620_s_atgb|AAD47191.1|AF106084_1 (AF106084) 4-coumarate: CoA ligase 1[Arabidopsis thaliana] ATU20347_S_AT ATU20347 15649_s_atgb|AAA91976.1|(U20347) mRNA corresponding to this gene accumulates inresponse to ATU21214_S_AT ATU21214 15590_s_at gb|AAA86507.1|(U21214)pyruvate dehydrogenase E1 alpha subunit [Arabidopsis thaliana]ATU21557_S_AT ATU21557 16098_s_at gb|AAC49255.1|(U21557) phosphoproteinphosphatase 2A, regulatory subunit A [Arabidopsis thaliana]ATU22340_S_AT ATU22340 15136_s_at gb|AAB49030.1|(U22340) DnaJ homolog[Arabidopsis thaliana] ATU36765_S_AT ATU36765 15177_s_atgb|AAC49079.1|(U36765) TGF-beta receptor interacting protein 1 homolog[Arabidopsis thaliana] ATU37235_S_AT ATU37235 15195_s_atemb|CAB58515.1|(A74281) unnamed protein product [Arabidopsis thaliana]ATU37281_F_AT ATU37281 16158_at gb|AAB52506.1|(U27811) actin7[Arabidopsis thaliana] ATU37587_S_AT ATU37587 13205_s_atgb|AAC49120.1|(U37587) cell division cycle protein [Arabidopsisthaliana] ATU39485_S_AT ATU39485 15122_s_at gb|AAC49281.1|(U39485) deltatonoplast integral protein [Arabidopsis thaliana] ATU43325_S_AT ATU4332515691_s_at gb|AAB39480.1|(U43325) profilin 1 [Arabidopsis thaliana]ATU43397_S_AT ATU43397 15112_s_at gb|AAD09837.1|(U43397) cryptochrome 2apoprotein [Arabidopsis thaliana] and cryptochrome 2 apoprotein (CRY2)(gb|U43397). ESTs gb|W43661 and gb|Z25638 come from this gene.[Arabidopsis thaliana] ATU46665_S_AT ATU46665 14730_s_atgb|AAC31617.1|(U49937) glutamate decarboxylase [Arabidopsis thaliana]Arabidopsis thaliana. ESTs gb|W43856, gb|N37724, gb|Z34642 and gb|R90491come from this gene. ATU49072_S_AT ATU49072 15215_s_atgb|AAB84353.1|(U49072) IAA16 [Arabidopsis thaliana] ATU49259_S_ATATU49259 15652_s_at gb|AAF26982.1|AC018363_27 (AC018363) isopentenyldiphosphate: dimethylallyl diphosphate isomerase [Arabidopsis thaliana]ATU52851_S_AT ATU52851 15197_s_at gb|AAB09723.1|(U52851) argininedecarboxylase [Arabidopsis thaliana] ATU56929_S_AT ATU56929 15180_s_atgb|AAB57799.1|(AF001535) AGAA.4 [Arabidopsis thaliana] ATU63633_S_ATATU63633 14721_s_at gb|AAB17665.1|(U63633) S- adenosylmethioninedecarboxylase [Arabidopsis thaliana] ATU66343_S_AT ATU66343 15654_s_atgb|AAC49695.1|(U66343) calreticulin [Arabidopsis thaliana] ATU68545_S_ATATU68545 14722_s_at gb|AAA74737.1|(U02565) 14-3-3- like protein 1[Arabidopsis thaliana] ATU75191_S_AT ATU75191 15216_s_atgb|AAB51576.1|(U75198) germin- like protein [Arabidopsis thaliana]ATU77381_S_AT ATU77381 16106_s_at gb|AAB82647.1|(U77381) WD-40 repeatprotein [Arabidopsis thaliana] ATU78297_F_AT ATU78297 15100_f_atgb|AAB36949.1|(U78297) plasma membrane intrinsic protein PIP3[Arabidopsis thaliana] ATU78870_S_AT ATU78870 17030_s_atgb|AAB68038.1|(U78866) gene 1000 [Arabidopsis thaliana] ATU79960_S_ATATU79960 16056_s_at gb|AAB72112.1|(U79960) vacuolar sorting receptorhomolog [Arabidopsis thaliana] ATU80186_S_AT ATU80186 15627_s_atgb|AAB86804.1|(U80186) pyruvate dehydrogenase E1 beta subunit[Arabidopsis thaliana] ATU91995_S_AT ATU91995 16170_s_atgb|AAD49755.1|AC007932_3 (AC007932) Identical to gb|U91995 Argonauteprotein from Arabidopsis CATL_S_AT CATL 13218_s_atgb|AAC17732.1|(AF021937) catalase 3 [Arabidopsis thaliana] CYSPROL_S_ATCYSPROL 13230_s_at emb|CAB10398.1|(Z97340) cysteine proteinase likeprotein [Arabidopsis thaliana] D01027.1_AT D01027.1 18940_atgb|AAC24370.1|(U89959) ARA-5 [Arabidopsis thaliana] D11394.4_S_ATD11394.4 16011_s_at emb|CAA44630.1|(X62818) Metallothionein-like protein[Arabidopsis thaliana] D13043.4_AT D13043.4 15973_atdbj|BAA02374.1|(D13043) thiol protease [Arabidopsis thaliana]D83531_S_AT D83531 15113_s_at dbj|BAA11944.1|(D83531) GDP dissociationinhibitor [Arabidopsis thaliana] D88374_S_AT D88374 15149_s_atdbj|BAA13599.1|(D88374) gamma subunit of mitochondrial F1-ATPase[Arabidopsis [Arabidopsis thaliana] GLUTATHIONEPEROXIDASE1_S_ATGLUTATHIONE 13259_s_at gb|AAD24836.1|AC007071_8 PEROXIDASE1 (AC007071)putative glutathione peroxidase [Arabidopsis thaliana] GST1_RC_S_AT GST113263_s_at emb|CAA10060.1|(AJ012571) glutathione transferase[Arabidopsis thaliana] GST2_S_AT GST2 13264_s_at emb|CAA72973.1|(Y12295)glutathione transferase [Arabidopsis thaliana] GST8_S_AT GST8 13267_s_atemb|CAA10060.1|(AJ012571) glutathione transferase [Arabidopsis thaliana]HSC701_S_AT HSC701 13269_s_at emb|CAA54419.1|(X77199) heat shock cognate70-1 [Arabidopsis thaliana] IAA16_S_AT IAA16 13294_s_atgb|AAB84353.1|(U49072) IAA16 [Arabidopsis thaliana] IAA8_S_AT IAA813663_s_at gb|AAD15575.1|(AC006340) auxin- regulated protein (IAA8)[Arabidopsis thaliana] J05216.2_S_AT J05216.2 16985_s_atgb|AAA32866.1|(J05216) ribosomal protein S11 (probable start codon at bp67) [Arabidopsis thaliana] L09755.2_S_AT L09755.2 19682_s_atgb|AAA32862.1|(L09755) ribosomal protein S28 [Arabidopsis thaliana]L14844_3_S_AT L14844 12824_s_at No hits found less than or equal to1e−15. L15389_S_AT L15389 18679_s_at No hits found. L26984_S_AT L2698418682_s_at gb|AAC27463.1|(AC003672) putative small GTP-binding protein[Arabidopsis thaliana] M21415.4_AT M21415.4 15988_atgb|AAA32757.1|(M21415) beta- tubulin [Arabidopsis thaliana] M55077.2_ATM55077.2 15993_at gb|AAA32868.1|(M55077) S- adenosylmethioninesynthetase [Arabidopsis thaliana] M64116_3_S_AT M64116 12827_s_atgb|AAA32794.1|(M64116) cystolic glyceraldehyde-3-phosphate dehydrogenase(GapC) [Arabidopsis thaliana] M84703.2_AT M84703.2 16480_atgb|AAA32884.1|(M84703) beta-6 tubulin [Arabidopsis thaliana] ORYZAIN4_ATORYZAIN4 14245_at dbj|BAA02374.1|(D13043) thiol protease [Arabidopsisthaliana] ORYZAIN5_AT ORYZAIN5 14246_at emb|CAA68192.1|(X99936) cysteineprotease [Zea mays] PHYA_AT PHYA 14622_at emb|CAA35221.1|(X17341) phyAphotoreceptor [Arabidopsis thaliana] RAN1_S_AT RAN1 14641_s_atgb|AAD29109.1|AF082565_1 (AF082565) ATP dependent copper transporter[Arabidopsis thaliana] RD19A_S_AT RD19A 14644_s_atemb|CAB38829.1|(AL035679) drought-inducible cysteine proteinase RD19Aprecursor S69727.2_AT S69727.2 16503_at gb|AAB20558.1|(S69727) light-regulated glutamine synthetase isoenzyme [Arabidopsis thaliana, Peptide,430 aa] THIOLPROTEASE1_S_AT THIOLPROTEASE1 14658_s_atemb|CAB38829.1|(AL035679) drought-inducible cysteine proteinase RD19Aprecursor [Arabidopsis thaliana] THIOLPROTEASE3_S_AT THIOLPROTEASE314659_s_at emb|CAB38829.1|(AL035679) drought-inducible cysteineproteinase RD19A precursor TONOL_F_AT TONOL 14662_f_atemb|CAA38633.1|(X54854) possible membrane channel protein [Arabidopsisthaliana] U11256.2_AT U11256.2 16035_at gb|AAA82212.1|(U11256)metallothionein [Arabidopsis thaliana] U15108.2_S_AT U15108.2 16010_s_atgb|AAA50250.1|(U15108) metallothionein-like protein [Arabidopsisthaliana] U20347.2_S_AT U20347.2 18651_s_at gb|AAA91976.1|(U20347) mRNAcorresponding to this gene accumulates in response to U21214_S_AT U2121418687_s_at gb|AAA86507.1|(U21214) pyruvate dehydrogenase E1 alphasubunit [Arabidopsis thaliana] U33014.2_S_AT U33014.2 15955_s_atgb|AAB53929.1|(U33014) polyubiquitin [Arabidopsis thaliana]U35640.2_S_AT U35640.2 16032_s_at gb|AAC49351.1|(U35640) thioredoxin h[Arabidopsis thaliana] U35826.2_S_AT U35826.2 19654_s_atgb|AAC49353.1|(U35826) thioredoxin h [Arabidopsis thaliana] U41998.4_ATU41998.4 16476_at gb|AAB37098.1|(U41998) actin 2 [Arabidopsis thaliana]U43224_S_AT U43224 12842_s_at No hits found less than or equal to 1e−15.U63815.18_S_AT U63815.18 16429_s_at gb|AAB07880.1|(U63815) ascorbateperoxidase [Arabidopsis thaliana] U64912.1_S_AT U64912.1 18989_s_atgb|AAB86892.1|(AF032883) AtJ3 [Arabidopsis thaliana] U65471_AT U6547118692_at No hits found less than or equal to 1e−15. U84969_3_F_AT U8496912833_f_at gb|AAB95252.1|(U84969) ubiquitin [Arabidopsis thaliana]U95973.108_AT U95973.108 18639_at gb|AAB65482.1|(U95973) endomembraneprotein EMP70 precusor isolog [Arabidopsis thaliana] WT108A_RC_AT WT108A14690_at No hits found less than or equal to 1e−15. WT755_S_AT WT75514701_s_at emb|CAA52237.1|(X74140) RCI14A [Arabidopsis thaliana]WT758_AT WT758 14703_at gb|AAD46040.1|AC007519_25 (AC007519) ESTsgb|H36253 and gb|AA04251 come from this gene. [Arabidopsis thaliana]X15550_S_AT X15550 12843_s_at gb|AAD26488.1|AC007195_2 (AC007195)unknown protein [Arabidopsis thaliana] X16432.2_S_AT X16432.2 15992_s_atemb|CAA34456.1|(X16432) elongation factor 1-alpha [Arabidopsis thaliana]X52256.2_AT X52256.2 16443_at emb|CAB45802.2|(AL080253) translationelongation factor EF-Tu precursor, chloroplast [Arabidopsis thaliana]X65052_AT X65052 16026_at emb|CAA46188.1|(X65052) eukaryotic translationinitiation factor 4A-1 [Arabidopsis thaliana] X65549.1_AT X65549.115963_at emb|CAA46518.1|(X65549) adenylate translocator [Arabidopsisthaliana] X68150.1_AT X68150.1 16451_at emb|CAA48253.1|(X68150) ketol-acid reductoisomerase [Arabidopsis thaliana] X69294.2_S_AT X69294.216030_s_at emb|CAA49155.1|(X69294) transmembrane protein TMP-B[Arabidopsis thaliana] X74604.2_S_AT X74604.2 15953_s_atemb|CAA52684.1|(X74604) heat shock protein 70 cognate [Arabidopsisthaliana] X74733.2_AT X74733.2 16463_at emb|CAA52751.1|(X74733)elongation factor-1 beta A1 [Arabidopsis thaliana] X75162.2_AT X75162.216997_at emb|CAA53005.1|(X75162) BBC1 protein [Arabidopsis thaliana]thaliana] X75881.2_AT X75881.2 16446_at emb|CAA53475.1|(X75881) plasmamembrane intrinsic protein 1a [Arabidopsis thaliana] X75883.2_ATX75883.2 15989_at emb|CAB67649.1|(AL132966) plasma membrane intrinsicprotein 2a [Arabidopsis thaliana] X78584.2_AT X78584.2 16456_atemb|CAA55321.1|(X78584) Di19 [Arabidopsis thaliana] X81697.2_S_ATX81697.2 16918_s_at emb|CAA57343.1|(X81697) cysteine synthase[Arabidopsis thaliana] X82002.1_AT X82002.1 20261_atemb|CAA57528.1|(X82002) protein phosphatase 2A 65 kDa regulatory subunit[Arabidopsis thaliana] X84078_AT X84078 18710_at emb|CAA58887.1|(X84078)NADH dehydrogenase [Arabidopsis thaliana] X84315.8_AT X84315.8 18659_atNo hits found less than or equal to 1e−15. X84318_AT X84318 18711_atemb|CAA59061.1|(X84318) NADH dehydrogenase [Arabidopsis thaliana]X86962.3_AT X86962.3 19917_at emb|CAA60525.1|(X86962) protein kinasecatalytic domain (fragment) [Arabidopsis thaliana] X91398.2_AT X91398.216988_at emb|CAA62744.1|(X91398) transcription factor L2 [Arabidopsisthaliana] X91958.1_AT X91958.1 16469_at emb|CAA63024.1|(X91958) 60Sribosomal protein L9 [Arabidopsis thaliana] X91959.1_AT X91959.115890_at gb|AAF04877.1|AC010796_13 (AC010796) 60S ribosomal protein L27A[Arabidopsis thaliana] X92510.2_S_AT X92510.2 19706_s_atemb|CAA63266.1|(X92510) allene oxide synthase [Arabidopsis thaliana]X94626.1_AT X94626.1 16508_at emb|CAA64329.1|(X94626) AATP2 [Arabidopsisthaliana] X99609.2_S_AT X99609.2 17430_s_at emb|CAA67923.1|(X99609)ubiquitin-like protein [Arabidopsis thaliana] Y07765.7_S_AT Y07765.716437_s_at No hits found less than or equal to 1e−15. Y09482.2_I_ATY09482.2 16036_i_at emb|CAA70691.1|(Y09482) HMG1 [Arabidopsis thaliana]Y10157.3_S_AT Y10157.3 19833_s_at emb|CAA71239.1|(Y10157) sulfitereductase [Arabidopsis thaliana] Y10863.1_I_AT Y10863.1 19919_i_atemb|CAA71879.1|(Y10986) hypothetical protein 194 [Arabidopsis thaliana]Y12295.2_S_AT Y12295.2 16033_s_at emb|CAA72973.1|(Y12295) glutathionetransferase [Arabidopsis thaliana] Y14052.2_AT Y14052.2 16506_atemb|CAA74381.1|(Y14052) ribosomal protein S6 [Arabidopsis thaliana]Y17053.2_AT Y17053.2 15960_at emb|CAA76606.1|(Y17053) At- hsc70-3[Arabidopsis thaliana] Z12024_AT Z12024 18731_at emb|CAA78059.1|(Z12024)calmodulin [Arabidopsis thaliana] Z14989.5_AT Z14989.5 17414_atemb|CAA78713.1|(Z14989) ubiquitin conjugating enzyme homolog[Arabidopsis thaliana] Z15157.1_AT Z15157.1 16982_atemb|CAA78856.1|(Z15157) Wilm's tumor suppressor homologue [Arabidopsisthaliana] Z28702.2_AT Z28702.2 16984_at emb|CAA82273.1|(Z28701) S18ribosomal protein [Arabidopsis thaliana] Z97335.5_S_AT Z97335.516504_s_at emb|CAB10172.1|(Z97335) hydroxymethyltransferase [Arabidopsisthaliana] Z97336.1_AT Z97336.1 16930_at emb|CAB10211.1|(Z97336)ribosomal protein [Arabidopsis thaliana] Z97337.298_S_AT Z97337.29816934_s_at emb|CAB10279.1|(Z97337) ribosomal protein [Arabidopsisthaliana] Z97340.298_S_AT Z97340.298 15972_s_at emb|CAB10398.1|(Z97340)cysteine proteinase like protein [Arabidopsis thaliana] Z97341.130_ATZ97341.130 18230_at emb|CAB10428.1|(Z97341) symbiosis-related likeprotein [Arabidopsis thaliana] Z97341.407_AT Z97341.407 18614_atemb|CAB10447.1|(Z97341) ribosomal protein [Arabidopsis thaliana]Z97343.270_S_AT Z97343.270 16926_s_at emb|CAB10520.1|(Z97343) ribosomalprotein [Arabidopsis thaliana] Z99708.65_AT Z99708.65 19139_atemb|CAB16820.1|(Z99708) ubiquitin—protein ligase-like protein[Arabidopsis thaliana]

Organ differential expressed genes were also analyzed. These genes wereexpressed at median level (average difference greater than 50) incertain organ at all developmental stages, e.g., compared to otherorgans, the expression level for these genes in the organ are 4-foldhigher than others. By these criteria, genes differentially expressed inroot (64) (see Table 5), leaf (94) (see Table 6), inflorescence stem(3), and flower (36) were identified, and functionally categorized.

TABLE 5 Arabidopsis sequences which are expressed in a root-specificmanner and their corresponding genes. Accession # Affy # DescriptionA71588.1 14015_s_at pir||T10626 reticuline oxidase homolog F21C20.190 -Arabidopsis thaliana >gi|5262224|emb|CAB45850.1| (AL080254) reticulineoxidase-like protein [Arabidopsisthaliana] >gi|7268880|emb|CAB79084.1|(AL161553) reticuline oxidase-likeprotein [Arabidopsis thaliana] A71596.1 14016_s_atgb|AAD25763.1|AC007060_21 (AC007060) Strong similarity to F19I3.2gi|3033375 putative berberine bridge enzyme from Arabidopsis thalianaBAC gb|AC004238. A71597.1 12079_s_at “gb|AAD25757.1|AC007060_15(AC007060) Strong similarity to F19I3.2 gi|3033375 putative berberinebridge enzyme from Arabidopsis thaliana BAC gb|AC004238. ESTs gb|F19886,gb|Z30784 and gb|Z30785 come from this gene” AB023448.2 12332_s_atdbj|BAA82824.1|(AB023462) basic endochitinase [Arabidopsis thaliana]AC001645.19 15965_at gb|AAC08601.1|(AF054906) myrosinase-binding proteinhomolog [Arabidopsis thaliana] AC001645.47 15996_atgb|AAB63635.1|(AC001645) jasmonate inducible protein isolog [Arabidopsisthaliana] AC001645.50 15981_at gb|AAB63635.1|(AC001645) jasmonateinducible protein isolog [Arabidopsis thaliana] AC002333.199 13552_atgb|AAB64044.1|(AC002333) putative endochitinase [Arabidopsis thaliana]AC002333.210 13154_s_at sp|Q06209|CHI4_BRANA BASIC ENDOCHITINASE CHB4PRECURSOR >gi|7435353|pir||S25311 chitinase (EC 3.2.1.14) precursor -rape >gi|17799|emb|CAA43708.1| (X61488) chitinase [Brassica napus]AC002391.150 17842_i_at pir||T04731 cytochrome P450 homolog F6G17.20 -Arabidopsis thaliana >gi|4468803|emb|CAB38204.1| (AL035601) cytochromeP450-like protein [Arabidopsisthaliana] >gi|7270719|emb|CAB80402.1|(AL161591) cytochrome P450-likeprotein [Arabidopsis thaliana] AC003673.201 16481_s_at pir||T01626peroxidase (EC 1.11.1.7) ATP22a - Arabidopsisthaliana >gi|3004558|gb|AAC09031.1|(AC003673) peroxidase (ATP22a)[Arabidopsis thaliana] AC004005.104 19390_at pir||T00681 hypotheticalprotein F6E13.14 - Arabidopsisthaliana >gi|3212858|gb|AAC23409.1|(AC004005) unknown protein[Arabidopsis thaliana] AC004521.114 19195_at pir||T02393 hypotheticalprotein F4I1.19 - Arabidopsisthaliana >gi|3128201|gb|AAC16105.1|(AC004521) unknown protein[Arabidopsis thaliana] AC004521.119 20608_s_at pir||T02393 hypotheticalprotein F4I1.19 - Arabidopsisthaliana >gi|3128201|gb|AAC1605.1|(AC004521) unknown protein[Arabidopsis thaliana] AC004683.79 16461_i_at sp|P24102|PERE_ARATH BASICPEROXIDASE E PRECURSOR >gi|81653|pir||JU0458 peroxidase (EC 1.11.1.7)E - Arabidopsis thaliana >gi|166807|gb|AAA32842.1|(M58381) peroxidase[Arabidopsis thaliana] AC004684.165 17907_s_at pir||T02541 hypotheticalprotein F13M22.25 - Arabidopsisthaliana >gi|3236257|gb|AAC23645.1|(AC004684) unknown protein[Arabidopsis thaliana] AC005310.6 17697_at pir||T02675 hypotheticalprotein F19D11.2 - Arabidopsisthaliana >gi|3510249|gb|AAC33493.1|(AC005310) unknown protein[Arabidopsis thaliana] AC005560.136 16016_at pir||G71401 probable majorlatex protein - Arabidopsis thaliana >gi|2244762|emb|CAB10185.1|(Z97335)major latex protein like [Arabidopsisthaliana] >gi|7268111|emb|CAB78448.1|(AL161538) major latex protein like[Arabidopsis thaliana] AC005560.147 12758_at pir||G71401 probable majorlatex protein - Arabidopsis thaliana >gi|2244762|emb|CAB10185.1|(Z97335)major latex protein like [Arabidopsisthaliana] >gi|7268111|emb|CAB78448.1|(AL161538) major latex protein like[Arabidopsis thaliana] AC005967.50 17864_at emb|CAA18195.1|(AL022198)putative protein [Arabidopsisthaliana] >gi|7270000|emb|CAB79816.1|(AL161578) putative protein[Arabidopsis thaliana] AC006216.22 14050_at gb|AAD12680.1|(AC006216)Similar to gi|3413714 T19L18.21 putative myrosinase-binding protein fromArabidopsis thaliana BAC gb|AC004747 AC006216.26 18571_at“gb|AAD12679.1|(AC006216) Similar to gi|3413714 T19L18.21 putativemyrosinase-binding protein from Arabidopsis thaliana BAC gb|AC004747.ESTs gb|T44298, gb|T42447, gb|R64761 and gb|I100206 come from this gene”AC006577.16 12778_r_at “gb|AAD25772.1|AC006577_8 (AC006577) Belongs tothe PF|00657 Lipase/Acylhydrolase with GDSL-motif family. ESTsgb|T44453, gb|T04815, gb|T45993, gb|R30138, gb|AI099570 and gb|T22281come from this gene. [Arabidopsis thaliana]” AC006587.164 15859_atgb|AAD21491.1|(AC006587) unknown protein [Arabidopsis thaliana]AC007060.34 19840_s_at gb|AAD25758.1|AC007060_16 (AC007060) Strongsimilarity to F19I3.2 gi|3033375 putative berberine bridge enzyme fromArabidopsis thaliana BAC gb|AC004238 AC007135.23 20176_atgb|AAD41993.1|AC006233_16 (AC006233) unknown protein [Arabidopsisthaliana] AC007584.48 20194_at gb|AAF20251.1|AC015450_12 (AC015450)unknown protein [Arabidopsis thaliana] ACHI 12852_s_atdbj|BAA21873.1|(AB006068) acidic endochitinase [Arabidopsis thaliana]AF098630.3 19118_s_at gb|AAD12259.1|(AF098631) putative cell wall-plasmamembrane disconnecting CLCT protein [Arabidopsis thaliana] AF128395.1220395_at “sp|P33154|PR1_ARATH PATHOGENESIS-RELATED PROTEIN 1 PRECURSOR(PR-1) >gi|322557|pir||JQ1693 pathogenesis- related protein 1 precursor,17.6K - Arabidopsis thaliana >gi|166861|gb|AAA32863.1|(M90508) PR-1-likeprotein [Arabidopsis thaliana] >gi|3810599|gb|AAC69381.1| (AC005398)pathogenesis-related PR-1-like protein [Arabidopsis thaliana]”AJ133036.5 15969_s_at sp|P24101|PERC_ARATH NEUTRAL PEROXIDASE CPRECURSOR >gi|81652|pir||JU0457 peroxidase (EC 1.11.1.7) C - Arabidopsisthaliana >gi|166827|gb|AAA32849.1| (M58380) peroxidase [Arabidopsisthaliana] >gi|6522555|emb|CAB61999.1|(AL132967) peroxidase [Arabidopsisthaliana] >gi|742247|prf||2009327A peroxidase [Arabidopsis thaliana]AL024486.185 16299_at sp|P42620|YQJG_ECOLI HYPOTHETICAL 37.4 KD PROTEININ EXUR-TDCC INTERGENIC REGION (O328) >gi|7465984|pir||C65099hypothetical 37.4 kD protein in exuR-tdcC intergenic region -Escherichia coli (strain K-12) >gi|606043|gb|AAA57906.1|(U18997)ORF_o328 [Escherichia coli] >gi|1789489|gb|AAC76137.1|(AE000392)putative transferase [Escherichia coli] AL035538.245 16514_atpir||T05635 hypothetical protein F20D10.200 - Arabidopsisthaliana >gi|4467114|emb|CAB37548.1|(AL035538) putative protein[Arabidopsis thaliana] >gi|7270791|emb|CAB80473.1|(AL161592) putativeprotein [Arabidopsis thaliana] AL049500.57 16914_s_atsp|P50700|OSL3_ARATH OSMOTIN-LIKE PROTEIN OSM34PRECURSOR >gi|1362001|pir||S57524 osmotin precursor - Arabidopsisthaliana >gi|887390|emb|CAA61411.1| (X89008) osmotin [Arabidopsisthaliana] AL049638.193 20029_at pir||T06615 hypothetical proteinF16J13.150 - Arabidopsis thaliana >gi|4586113|emb|CAB40949.1|(AL049638)putative DNA-binding protein [Arabidopsisthaliana] >gi|7267909|emb|CAB78251.1|(AL161533) putative DNA- bindingprotein [Arabidopsis thaliana] AL049730.104 18983_s_at “pir||S42552proline-rich protein - rape >gi|545029|gb|AAC60566.1|(S68113)proline-rich SAC51 [Brassica napus = oilseed rape, pods, Peptide, 147aa]” AL080253.32 19415_at gb|AAF08575.1|AC011623_8 (AC011623) unknownprotein [Arabidopsis thaliana] AL080282.74 18597_at pir||T10624reticuline oxidase homolog F21C20.170 - Arabidopsisthaliana >gi|5262222|emb|CAB45848.1| (AL080254) reticuline oxidase-likeprotein [Arabidopsis thaliana] >gi|7268878|emb|CAB79082.1|(AL161553)reticuline oxidase-like protein [Arabidopsis thaliana] ATAJ259616085_s_at emb|CAB16787.1|(Z99707) patatin-like protein [Arabidopsisthaliana] >gi|7270656|emb|CAB80373.1|(AL161590) patatin-like protein[Arabidopsis thaliana] ATHORF 16649_s_at gb|AAF16563.1|AC012563_16(AC012563) putative S- adenosyl-L-methionine: trans-caffeoyl-Coenzyme A3-O- methyltransferase [Arabidopsis thaliana] ATPIN2 12932_s_atgb|AAD04377.1|(AF089085) putative auxin efflux carrier protein; AtPIN1[Arabidopsis thaliana] ATU10034 15120_s_at sp|Q42521|DCE1_ARATHGLUTAMATE DECARBOXYLASE 1 (GAD 1) >gi|497979|gb|AAA93132.1|(U10034)glutamate decarboxylase [Arabidopsis thaliana] ATU57320 15137_s_atgb|AAB47973.1|(U57320) blue copper-binding protein II [Arabidopsisthaliana] ATU62330 15623_f_at dbj|BAA24282.1|(AB000094) inorganicphosphate transporter [Arabidopsis thaliana] BCHI 13211_s_atdbj|BAA82824.1|(AB023462) basic endochitinase [Arabidopsis thaliana]CAFFEROYLCOA - 13215_s_at gb|AAA62426.1|(L40031)S-adenosyl-L-methionine: trans- METHYLTRANS caffeoyl-Coenzyme A3-O-methyltransferase [Arabidopsis thaliana] NOVARTIS51 14170_atgb|AAF29406.1|AC022354_5 (AC022354) unknown protein [Arabidopsisthaliana] U72155.2 15954_at gb|AAB64244.1|(U72155) beta-glucosidase[Arabidopsis thaliana] U81294.2 20422_g_at gb|AAD00509.1|(U81294)germin-like protein [Arabidopsis thaliana] X67421.3 16489_at pir||S53012root-specific protein RCc3 - rice >gi|786132|gb|AAA65513.1|(L27208) RCc3[Oryza sativa] X74514.2 20239_g_at dbj|BAA89048.1|(AB029310)beta-fructofuranosidase [Arabidopsis thaliana] X78586.2 16048_atpir||S51480 drought-induced protein Dr4 - Arabidopsisthaliana >gi|469114|emb|CAA55323.1|(X78586) Dr4 [Arabidopsis thaliana]X98319.2 16971_s_at emb|CAA66963.1|(X98319) peroxidase [Arabidopsisthaliana] >gi|1429217|emb|CAA67311.1|(X98775) peroxidase ATP12a[Arabidopsis thaliana] >gi|6714469|gb|AAF26155.1|AC008261_12 (AC008261)putative peroxidase [Arabidopsis thaliana] X98320.2 18312_s_atgb|AAF63027.1|AF244924_1 (AF244924) peroxidase prx15 precursor [Spinaciaoleracea] X98321.2 19595_s_at gb|AAB71452.1|(AC000098) Strong similarityto Arabidopsis peroxidase ATPEROX7A (gb|X98321). [Arabidopsisthaliana] >gi|2738254|gb|AAB94661.1|(U97684) peroxidase precursor[Arabidopsis thaliana] X98322.2 17942_s_at gb|AAF03466.1|AC009327_5(AC009327) putative peroxidase [Arabidopsis thaliana] X98808.1 15985_atemb|CAA67340.1|(X98808) peroxidase ATP3a [Arabidopsis thaliana] X98855.216028_at pir||T01626 peroxidase (EC 1.11.1.7) ATP22a - Arabidopsisthaliana >gi|3004558|gb|AAC09031.1|(AC003673) peroxidase (ATP22a)[Arabidopsis thaliana] Y11788.1 18946_at emb|CAA72484.1|(Y11788)peroxidase ATP24a [Arabidopsis thaliana] Z97338.321 16045_s_atpir||E71418 hypothetical protein - Arabidopsisthaliana >gi|2244897|emb|CAB10319.1|(Z97338) HSR201 like protein[Arabidopsis thaliana] >gi|7268287|emb|CAB78582.1|(AL161541) HSR201 likeprotein [Arabidopsis thaliana] Z97340.345 17485_s_at“sp|P52407|E13B_HEVBR GLUCAN ENDO-1,3-BETA- GLUCOSIDASE, BASIC VACUOLARISOFORM PRECURSOR ((1- >3)-BETA-GLUCAN ENDOHYDROLASE) ((1->3)-BETA-GLUCANASE) (BETA-1,3-ENDOGLUCANASE) >gi|2129912|pir||S650771,3-beta-glucanase (EC 3.2.1.—) precursor - Para rubbertree >gi|1184668|gb|AAA87456.1| (U22147) beta-1,3-glucanase [Heveabrasiliensis]” Z97344.151 19886_at gb|AAC61811.1|(AC004667) putativeAT-hook DNA-binding protein [Arabidopsis thaliana] Z99707.288 18326_s_atemb|CAB16788.1|(Z99707) patatin-like protein [Arabidopsisthaliana] >gi|7270655|emb|CAB80372.1|(AL161590) patatin-like protein[Arabidopsis thaliana]

TABLE 6 Arabidopsis sequences which are expressed in a leaf-specificmanner and their corresponding genes. Affy ID Accession functionDescription 11994_at AC004218.86_AT novel gb|AAC27838.1|(AC004218)unknown protein [Arabidopsis thaliana] 12086_s_at AC002409.88_S_AT novelgb|AAB86456.1|(AC002409) unknown protein [Arabidopsis thaliana] 12095_atAC006223.95_AT novel gb|AAD15394.1|(AC006223) hypothetical protein[Arabidopsis thaliana] 12105_at AF000657.30_AT novelgb|AAB72170.1|(AF000657) hypothetical protein [Arabidopsis thaliana]12115_at AL033545.26_AT metabolism emb|CAA22152.1|(AL033545)extensin-like protein [Arabidopsis thaliana] 12135_at AC007230.29_ATnovel gb|AAD26875.1|AC007230_9 (AC007230) ESTs gb|H76289 and gb|H76537come from this gene. [Arabidopsis thaliana] 12270_at AL030978.79_ATkinase emb|CAA19724.1|(AL030978) putative receptor protein kinase[Arabidopsis thaliana] 12299_at AL022347.265_AT kinaseemb|CAA18476.1|(AL022347) serine/threonine kinase-like protein[Arabidopsis thaliana] 12305_i_at AL022347.219_I_AT novelemb|CAA18473.1|(AL022347) putative protein [Arabidopsis thaliana]12392_at AC002391.102_AT transcription gb|AAB87103.1|(AC002391) putativeMYB family transcription factor [Arabidopsis thaliana] 12788_atAC002311.20_AT defense “gb|AAC00607.1|(AC002311) similar toripening-induced protein, gp|AJ001449|2465015 and major#latex protein,gp|X91961|1107495 [Arabidopsis thaliana]” 13243_r_at ELI32_R_ATmetabolism emb|CAB37539.1|(AL035538) cinnamyl-alcohol dehydrogenaseELI3-2 [Arabidopsis 13352_at AL030978.126_AT novelemb|CAA19730.1|(AL030978) putative protein [Arabidopsis thaliana]13620_at AL035605.41_AT metabolism emb|CAB38295.1|(AL035605)formamidase-like protein [Arabidopsis thaliana] 13719_at NOVARTIS106_ATnovel No hits found less than or equal to 1e−15. 13812_s_atAC005275.104_S_AT hormone gb|AAD14468.1|(AC005275) putative GH3-likeprotein [Arabidopsis thaliana] 13972_s_at Z97344.134_S_AT transcriptionemb|CAB10561.1|(Z97344) SUPERMAN like protein [Arabidopsis thaliana]14192_at NOVARTIS66_AT novel gb|AAC34331.1|(AC004122) Unknown protein[Arabidopsis thaliana] 14218_at NOVARTIS87_AT novel No hits found lessthan or equal to 1e−15. 14242_s_at NRA_S_AT metabolismgb|AAF19225.1|AC007505_1 (AC007505) nitrate reductase [Arabidopsisthaliana] 14248_at PAD3_AT metabolism “gb|AAD31062.1|AC007357_11(AC007357) Strong similarity to gb|X97864 cytochrome P450 fromArabidopsis thaliana and is a member of the PF|00067 Cytochrome P450family. ESTs gb|N65665, gb|T14112, gb|T76255, gb|T20906 and gb|AI100027come from this gene.” 14432_at AL035440.502_AT novelemb|CAB36549.1|(AL035440) putative protein [Arabidopsis thaliana]14484_at U73462.2_AT metabolism gb|AAC32523.1|(U73462) carbonicanhydrase [Arabidopsis thaliana] 14533_i_at AC007048.166_I_AT novelgb|AAC32523.1|(U73462) carbonic anhydrase [Arabidopsis thaliana]14600_at AC007576.49_AT novel gb|AAD39297.1|AC007576_20 (AC007576)Unknown protein [Arabidopsis thaliana] 14603_at AL022347.282_AT kinaseemb|CAA18477.1|(AL022347) serine/threonine kinase-like protein[Arabidopsis thaliana] 14621_at PDF1.2_AT defensegb|AAC31244.1|(AC004747) putative antifungal protein [Arabidopsisthaliana] 14635_s_at PR.1_S_AT defense gb|AAC69381.1|(AC005398)pathogenesis-related PR-1-like protein [Arabidopsis thaliana] 14682_i_atWT1012A_RC_I_AT novel No hits found. 14709_at WT788_AT novel No hitsfound less than or equal to 1e−15. 14803_at AC006550.33_AT metabolismgb|AAD25807.1|AC006550_15 (AC006550) Strong similarity to gb|Z49699glutaredoxin from Ricinus communis. [Arabidopsis thaliana] 14808_i_atAC007230.21_I_AT kinase gb|AAD26873.1|AC007230_7 (AC007230) ContainsPF|00069 Eukaryotic protein kinase domain. [Arabidopsis thaliana]14862_at AC005770.205_AT transcription gb|AAC79620.1|(AC005770) putativeRING zinc finger protein [Arabidopsis thaliana] 15185_s_at AB024283_S_ATmetabolism dbj|BAA78561.1|(AB024283) cysteine synthase [Arabidopsisthaliana] 15271_at AC004077.141_AT novel gb|AAC26689.1|(AC004077)unknown protein [Arabidopsis thaliana] 15422_at AF069441.29_AT novelgb|AAD36948.1|AF069441_8 (AF069441) hypothetical protein [Arabidopsisthaliana] 15467_at AC000375.34_AT novel gb|AAB60770.1|(AC000375) ESTgb|H37044 comes from this gene. [Arabidopsis thaliana] 15552_atAL096859.162_AT novel emb|CAB51187.1|(AL096859) putative protein[Arabidopsis thaliana] 15613_s_at ATHHOMEOA_S_AT metabolismemb|CAA79670.1|(Z19602) HAT4 [Arabidopsis thaliana] 15837_atAC005496.175_AT metabolism gb|AAC35232.1|(AC005496) putative thiaminbiosynthesis protein [Arabidopsis thaliana] 16137_s_at AF149053_S_ATmetabolism gb|AAD38033.1|AF149053_1 (AF149053) phytochrome kinasesubstrate 1 [Arabidopsis thaliana] 16172_s_at D78603_S_AT metabolismdbj|BAA28535.1|(D78603) cytochrome P450 monooxygenase [Arabidopsisthaliana] 16322_at AL096860.203_AT novel emb|CAB51215.1|(AL096860)putative protein [Arabidopsis thaliana] 16323_at AC005957.35_AT defensegb|AAD03365.1|(AC005957) putative disease resistance protein[Arabidopsis thaliana] 16331_at AC005957.23_AT defensegb|AAD03361.1|(AC005957) putative disease resistance protein[Arabidopsis thaliana] 16365_at AC003974.136_AT defensegb|AAC04495.1|(AC003974) putative disease resistance protein[Arabidopsis thaliana] 16547_s_at AF053941_S_AT metabolismgb|AAC27293.2|(AF053941) non phototropic hypocotyl 1-like [Arabidopsisthaliana] 16583_s_at ATHZFPH_S_AT transcription gb|AAA87304.1|(L39651)zinc finger protein [Arabidopsis thaliana] 16687_s_at AC004044.64_S_ATnovel gb|AAC79114.1|(AF069442) hypothetical protein [Arabidopsisthaliana] 16845_at AC006232.87_AT metabolism gb|AAD15594.1|(AC006232)putative cysteine proteinase [Arabidopsis thaliana] 16856_i_atAC004681.86_I_AT metabolism gb|AAC25936.1|(AC004681) putative cellulosesynthase [Arabidopsis thaliana] 17019_s_at ATU28422_S_AT transcriptiongb|AAC33507.1|(AC005310) MYB- related transcription factor (CCA1)[Arabidopsis thaliana] 17128_s_at ATHRPRP1A_S_AT defensegb|AAC69381.1|(AC005398) pathogenesis-related PR-1-like protein[Arabidopsis 17231_at AC004411.170_AT novel gb|AAC34226.1|(AC004411)hypothetical protein [Arabidopsis thaliana] 17331_at AF069298.23_ATkinase “gb|AAC19274.1|(AF069298) contains similarity to a protein kinasedomain (Pfam: pkinase.hmm, score: 165.48), to legume lectins beta domain(Pfam: lectin_legB.hmm, score: 125.64) and legume lectins alpha domain(Pfam: lectin_legA.hmm, score: 16.72) [Arabi 17361_s_at AF096373.28_S_ATmetabolism emb|CAB39764.1|(AL049487) sucrose-phosphate synthase-likeprotein [Arabidopsis thaliana] 17411_s_at X98926.1_S_AT defenseemb|CAA67426.1|(X98926) thylakoid-bound ascorbate peroxidase[Arabidopsis thaliana] 17815_s_at Z97342.284_S_AT defenseemb|CAB46050.1|(Z97342) disease resistance RPP5 like protein (fragment)[Arabidopsis thaliana] 17835_at AF096370.14_AT RNA bindinggb|AAC62779.1|(AF096370) protein contains similarity to Arabidopsisthaliana reverse transcriptase-like proteins 17861_s_at AC005560.16_S_ATtransport gb|AAC67319.1|(AC005560) putative auxin transport protein[Arabidopsis thaliana] 17936_s_at Z97342.384_S_AT metabolismemb|CAB46051.1|(Z97342) putative beta-amylase [Arabidopsis thaliana]18115_at AC005388.43_AT kinase gb|AAC64891.1|(AC005388) Similar toT11J7.13 gi|2880051 putative protein kinase from Arabidopsis thalianaBAC gb|AC002340. 18296_at AC002510.60_AT kinase gb|AAB84338.1|(AC002510)putative Ca2+-ATPase [Arabidopsis thaliana] 18301_s_at AL022223.48_S_ATmetabolism emb|CAA18218.1|(AL022223) fructose-bisphosphate aldolase[Arabidopsis thaliana] 18469_at AC006341.12_AT kinasegb|AAD34678.1|AC006341_6 (AC006341) Similar to gb|AJ012423wall-associated kinase 2 from Arabidopsis thaliana. 18588_atAL022604.205_AT novel emb|CAA18744.1|(AL022604) putative protein[Arabidopsis thaliana] 18670_g_at AJ250341_G_AT metabolismemb|CAB58423.1|(AJ250341) beta- amylase enzyme [Arabidopsis thaliana]18778_at Z97338.384_AT novel emb|CAB10322.1|(Z97338) hypotheticalprotein [Arabidopsis thaliana] 18811_at AC002396.32_AT novelgb|AAC00583.1|(AC002396) Hypothetical protein [Arabidopsis thaliana]18835_at AC007260.34_AT novel gb|AAD30584.1|AC007260_15 (AC007260)lcl|prt_seq No definition line found [Arabidopsis thaliana] 18844_atAC005315.131_AT transport gb|AAC33239.1|(AC005315) putative ligand-gatedion channel protein [Arabidopsis thaliana] 18866_at AC005917.178_ATtransposable gb|AAD10163.1|(AC005917) element putative Ta11-like non-LTRretroelement protein [Arabidopsis thaliana] 19034_at AL021768.117_ATdefense emb|CAA16930.1|(AL021768) TMV resistance protein N-like[Arabidopsis thaliana] 19465_at AL021768.96_AT defenseemb|CAA16929.1|(AL021768) resistance protein RPP5-like [Arabidopsisthaliana] 19581_at AC006526.102_AT transport gb|AAD23055.1|AC006526_14(AC006526) putative cyclic nucleotide-regulated ion channel protein[Arabidopsis thaliana] 19704_i_at AJ005927.2_I_AT metabolismemb|CAA06769.1|(AJ005927) squalene epoxidase homologue [Arabidopsisthaliana] 19718_at AC000098.16_AT transport gb|AAB71447.1|(AC000098)Similar to Arabidopsis Fe(II) transport protein (gb|U27590).[Arabidopsis thaliana] 19720_at AC003979.28_AT hormonegb|AAC25517.1|(AC003979) Contains similarity to gibberellin- regulatedprotein 2 precursor (GAST1) homolog gb|U11765 from A. thaliana.[Arabidopsis thaliana] 19774_at AC007167.248_AT transportgb|AAD30549.1|AF136580_1 (AF136580) iron-regulated transporter 2[Lycopersicon esculentum] 19834_at AC006264.14_AT hormonegb|AAD29795.1|AC006264_3 (AC006264) putative auxin-regulated protein[Arabidopsis thaliana] 19889_at AC003033.139_AT novelgb|AAB91986.1|(AC003033) unknown protein [Arabidopsis thaliana] 19901_atAC003033.129_AT novel gb|AAB91985.1|(AC003033) unknown protein[Arabidopsis thaliana] 19992_at AC007138.58_AT novelgb|AAD22657.1|AC007138_21 (AC007138) predicted protein of unknownfunction [Arabidopsis thaliana] 20062_at AC005896.23_AT novelgb|AAC98045.1|(AC005896) unknown protein [Arabidopsis thaliana] 20063_atAC006284.5_AT metabolism gb|AAD17422.1|(AC006284) putative esterase[Arabidopsis thaliana] 20232_s_at AL022347.12_S_AT kinaseemb|CAA18460.1|(AL022347) protein kinase-like protein [Arabidopsisthaliana] 20356_at AC004561.74_AT metabolism gb|AAC9519.1|(AC004561)putative glutathione S-transferase [Arabidopsis thaliana] 20429_s_atZ97336.167_S_AT novel emb|CAB10219.1|(Z97336) hypothetical protei[Arabidopsis thaliana] 20525_at AC007169.89_AT transcriptiongb|AAD26481.1|AC007169_13 (AC007169) putative CONSTANS-like B-box zincfinger protein [Arabidopsis thaliana] 20537_at AL049608.65_AT metabolismemb|CAB40769.1|(AL049608) extensin-like protein [Arabidopsis thaliana]20544_at AL035679.68_AT transcription emb|CAB38816.1|(AL035679) putativezinc finger protein [Arabidopsis thaliana] 20705_at AL049607.66_ATmetabolism emb|CAB40757.1|(AL049607) glutathione peroxidase-like protein[Arabidopsis thaliana]

To examine the organ-specificity of the differential expression, theexpression level of differentially expressed genes were plotted againstrepresented samples. The root differential expressed genes are expressedalmost exclusively in root and young whole seedlings. There were 51genes that were expressed only in root. Twenty-three percent of thesegenes had no known function while peroxidases and defense genesrepresented 51% of the genes.

Similar experiments were conducted for root at least 3 hours afterexposure to stress, e.g., salt, mannitol or cold (Tables 7-8).Twenty-five root-specific promoters were downregulated and 8 wereupregulated in response to salt stress, 21 were down-regulated and 17were upregulated in response to mannitol, and 22 were downregulated and7 were upregulated in response to cold. Ten promoters did not respond toany of the stresses.

Expression results from an acute (3 hour) response to stress, either upor down, to cold, mannitol, or salt in roots but not in leaves are shownbelow in Table 7. Of the nine root-specific promoters identified, onedid not show a response to any of the stresses, two were downregulatedin response to cold, mannitol and stress, four were upregulated inresponse to at least one of the stresses and downregulated in responseto at least one of the stresses, and two were only downregulated by saltstress.

TABLE 7 Cold Cold Man Man Salt Salt Accession Affy id Root3 Root27 Root3Root27 Root3 Root27 Roots AC006577.16 12778_r_at −1985 −3753 −2768 −363−4018 −1769 ATU57320 15137_s_at −729 −219 −1304 992 −2420 141 X98808.115985_at −2123 1183 −1881 −312 −2331 −343 U81294.2 20421_at −19 2399−1162 345 −1450 371 Z97338.321 16045_s_at −1068 −694 −1084 124 −1425−285 X98855.2 16028_at −448 −691 −595 −589 −1043 −559 AC006577.1612779_f_at −672 −763 −636 −419 −976 −559 X78586.2 16048_at 56 603 −576307 −881 −588 ATU62330 15623_f_at −1274 373 −1054 141 −817 439NOVARTIS51 14170_at −1058 537 −654 −14 −718 16 AC005560.136 16016_at 93643 25 628 −648 −232 AF098630.3 19118_s_at 228 422 −52 −37 −640 −117AF128395.12 20395_at −286 −508 −482 −115 −621 261 Z97340.345 17485_s_at−691 −1934 −357 −592 −529 −454 AL035538.245 16514_at 200 −498 798 935−490 −118 X98322.2 17942_s_at −366 54 −285 4 −457 3 ATU10034 15120_s_at−102 134 −336 −80 −456 −65 AL049730.104 18983_s_at 322 −51 −272 −167−439 −570 AJ133036.5 15969_s_at −316 −619 74 −465 −400 −470 U72155.215954_at 52 −178 −86 −447 −388 −252 X98319.2 16971_s_at −368 9 −291 −62−368 −86 U81294.2 20422_g_at −96 530 −272 43 −341 32 X67421.3 16489_at446 200 −158 −41 −323 −357 Y11788.1 18946_at 100 146 −58 −21 −199 124ATPIN2 12932_s_at −172 −182 −158 −67 −170 −128 AC005310.6 17697_at −9918 −97 −15 −139 −23 AC007135.23 20176_at −37 82 260 137 −120 −81AC006587.164 15859_at 91 134 29 13 −117 −8 AC004521.114 19195_at −410 93−322 −36 −96 −20 X98321.2 19595_s_at −50 −149 −66 0 −95 73 AC002333.19913552_at −205 −418 167 101 −89 −148 AL024486.185 16299_at −162 −165 −76−47 −80 −20 AC004521.119 20608_s_at −201 96 −119 −7 −75 15 A71597.112079_s_at −185 −153 79 −142 −74 −60 AC006216.26 18571_at −46 55 23 −26−71 10 AC006216.22 14050_at −45 14 −23 −14 −62 −8 AL080253.32 19415_at112 −132 107 118 −56 −108 AC004683.79 16461_i_at −145 −621 −136 −164 −17142 X74514.2 20239_g_at 13 213 60 −91 1 1 AL080282.74 18597_at −251 161−58 120 4 −24 AC002333.210 13153_r_at −5 −186 48 −82 9 −51 X74514.220238_at 288 553 174 115 10 302 CAFFEROYLCOA- 13215_s_at 42 33 38 −20 12−56 METHYLTRANS AC004005.104 19390_at −77 0 −121 37 13 −16 ATHORF16649_s_at 54 112 43 17 16 −8 AC003673.201 16481_s_at −38 −106 16 −22 17−28 ATAJ2596 16085_s_at 128 −137 240 64 30 −47 AC002333.210 13154_s_at−6 −511 168 −224 31 −172 AC004684.165 17907_s_at −154 −52 −3 106 40 65AL049638.193 20029_at 45 41 35 −42 64 −20 A71588.1 14015_s_at −130 138164 −23 79 −1 A71596.1 14016_s_at −104 99 132 −15 98 1 Z99707.28818326_s_at 150 −110 309 19 99 −75 ACHI 12852_s_at −25 36 97 −7 114 −20AC005560.147 12758_at 33 −822 362 357 121 146 X98320.2 18312_s_at 38 29293 21 131 −14 AC002391.150 17843_s_at 79 170 26 15 177 1 AC005967.5017864_at 37 133 41 −37 196 −4 AC007060.34 19840_s_at 606 1194 304 −145286 185 BCHI 13211_s_at 99 −554 337 −242 312 −275 AC001645.19 15965_at−323 −177 141 −437 355 −389 AB023448.2 12332_s_at 170 −704 421 −130 370−374 AC001645.47 15996_at −160 −167 215 −162 445 −147 AL049500.5716914_s_at 96 −2596 366 −818 541 −1265 AC007584.48 20194_at 288 0 848259 1016 −116 Cold Cold Man Man Salt Salt Accession Affy id Leaf3 Leaf27Leaf3 Leaf27 Leaf3 Leaf27 Leaves AC006577.16 12778_r_at 80 −89 92 −81−14 −167 ATU57320 15137_s_at 158 63 53 5 −35 −79 X98808.1 15985_at −5−136 −11 −137 5 −93 U81294.2 20421_at 35 −8 18 81 52 −19 Z97338.32116045_s_at 10 −8 1 2 5 −4 X98855.2 16028_at −1 −16 −2 −13 1 −13AC006577.16 12779_f_at −83 −57 −47 −53 −34 −58 X78586.2 16048_at 69 96149 78 36 81 ATU62330 15623_f_at −3 8 −4 42 49 −14 NOVARTIS51 14170_at−188 1031 −258 −311 −310 −195 AC005560.136 16016_at 1 0 7 7 4 5AF098630.3 19118_s_at 1 −9 −6 1 −2 −5 AF128395.12 20395_at 3 1 10 3 6 −2Z97340.345 17485_s_at 103 −619 20 −200 −54 −521 AL035538.245 16514_at 1510 6 10 5 −2 X98322.2 17942_s_at −1 0 −2 −2 2 −1 ATU10034 15120_s_at 10−85 −3 −81 −3 −25 Cold Cold Man Man Salt Salt Accession Affy id Root3Root27 Root3 Root27 Root3 Root27 AL049730.104 18983_s_at −6 13 0 14 −4 7AJ133036.5 15969_s_at 4 13 12 13 25 7 U72155.2 15954_at 4 4 0 −7 4 −2X98319.2 16971_s_at −4 3 3 −2 1 −5 U81294.2 20422_g_at 12 0 6 9 11 −4X67421.3 16489_at −3 2 −5 0 −2 2 Y11788.1 18946_at −177 −203 −175 −204−158 285 ATPIN2 12932_s_at −13 −1 −2 1 −3 −6 AC005310.6 17697_at −3 2 −1−3 0 −5 AC007135.23 20176_at 8 3 0 −1 1 −6 AC006587.164 15859_at −51 −62−54 −47 −56 50 AC004521.114 19195_at −35 2 −12 1 −3 −21 X98321.219595_s_at 2 −4 −1 0 0 2 AC002333.199 13552_at 4 7 −1 2 1 6 AL024486.18516299_at −15 −139 −26 −33 −31 −35 AC004521.119 20608_s_at −18 1 −15 −2 2−6 A71597.1 12079_s_at −4 −22 −5 −10 5 −7 AC006216.26 18571_at −1 9 2 104 10 AC006216.22 14050_at −2 −1 −3 −4 −2 2 AL080253.32 19415_at 6 0 3 02 6 AC004683.79 16461_i_at 26 0 8 17 14 21 X74514.2 20239_g_at −11 84 4−60 −55 −48 AL080282.74 18597_at −62 284 27 36 −40 23 AC002333.21013153_r_at 52 −23 41 35 −6 −42 X74514.2 20238_at −9 218 0 −112 −180 −194CAFFEROYLCOA- 13215_s_at 20 31 7 0 1 −8 METHYLTRANS AC004005.10419390_at 8 −3 −3 1 4 −13 ATHORF 16649_s_at 47 39 9 2 −2 −8 AC003673.20116481_s_at 3 0 0 5 1 7 ATAJ2596 16085_s_at 0 −1 −9 2 −3 1 AC002333.21013154_s_at 74 −63 198 75 −20 −84 AC004684.165 17907_s_at 17 −29 16 25 15−8 AL049638.193 20029_at −4 −18 −6 −5 0 −9 A71588.1 14015_s_at 5 −7 2 −613 −10 A71596.1 14016_s_at 8 −3 11 −2 −1 1 Z99707.288 18326_s_at 1 2 −13 0 −3 ACHI 12852_s_at 16 −6 9 9 8 −10 AC005560.147 12758_at 2 1 1 10 33 X98320.2 18312_s_at 1 −2 1 5 −2 0 AC002391.150 17843_s_at 416 −53 487239 184 63 AC005967.50 17864_at 8 8 5 10 5 0 AC007060.34 19840_s_at −80169 106 105 −2 50 BCHI 13211_s_at 44 −94 −1 −13 37 −54 AC001645.1915965_at −24 −3 −22 −4 25 −27 AB023448.2 12332_s_at 127 −172 9 −10 9−133 AC001645.47 15996_at 5 −10 6 −6 29 −20 AL049500.57 16914_s_at 265−341 19 −7 78 −354 AC007584.48 20194_at 27 182 78 62 30 32

TABLE 8 Root genes up- or down-regulated in response to cold, mannitolor salt stress Accession # Affy # Description Down regulated with coldstress in root (acute response 3 hrs) X98808.1 15985_atemb|CAA67340.1|(X98808) peroxidase ATP3a [Arabidopsis thaliana]AC006577.16 12778_r_at “gb|AAD25772.1|AC006577_8 (AC006577) Belongs tothe PF|00657 Lipase/Acylhydrolase with GDSL-motif family. ESTsgb|T44453, gb|T04815, gb|T45993, gb|R30138, gb|AI099570 and gb|T22281come from this gene. [Arabidopsis thaliana]” ATU62330 15623_f_atdbj|BAA21503.1|(D86591) inorganic phosphate transporter [Arabidopsisthaliana] Z97338.321 16045_s_at emb|CAB10318.1|(Z97338) HSR201 likeprotein [Arabidopsis thaliana] AC006577.16 12779_f_at“gb|AAD25772.1|AC006577_8 (AC006577) Belongs to the PF|00657Lipase/Acylhydrolase with GDSL-motif family. ESTs gb|T44453, gb|T04815,gb|T45993, gb|R30138, gb|AI099570 and gb|T22281 come from this gene.[Arabidopsis thaliana]” X98855.2 16028_at emb|CAA67361.1|(X98855)peroxidase ATP8a [Arabidopsis thaliana] AC004521.114 19195_atgb|AAC16105.1|(AC004521) unknown protein [Arabidopsis thaliana] X98319.216971_s_at emb|CAA66963.1|(X98319) peroxidase [Arabidopsis thaliana]X98322.2 17942_s_at emb|CAA66966.1|(X98322) peroxidase [Arabidopsisthaliana] AC001645.19 15965_at gb|AAB63631.1|(AC001645) jasmonateinducible protein isolog [Arabidopsis thaliana] AJ133036.5 15969_s_atemb|CAA67313.1|(X98777) peroxidase ATP16a [Arabidopsis thaliana]AF128395.12 20395_at “gb|AAD17355.1|(AF128395) contains similarity topathogenesis-related protein 1 precursors and SCP-like extracellularproteins (Pfam: PF00188, Score = 79.8, E = 4.1e−21, N = 1) [Arabidopsisthaliana]” AL080282.74 18597_at emb|CAB45881.1|(AL080282) berberinebridge enzyme- like protein [Arabidopsis thaliana] AC002333.199 13552_atgb|AAB64045.1|(AC002333) endochitinase isolog [Arabidopsis thaliana]AC004521.119 20608_s_at gb|AAC16106.1|(AC004521) hypothetical protein[Arabidopsis thaliana] A71597.1 12079_s_at emb|CAB42613.1|(A71641)unnamed protein product [Arabidopsis thaliana] ATPIN2 12932_s_atgb|AAC84042.1|(AF087459) polar-auxin-transport efflux componentAGRAVITROPIC 1 [Arabidopsis thaliana] AL024486.185 16299_atemb|CAA19705.1|(AL024486) putative protein [Arabidopsis thaliana]AC001645.47 15996_at gb|AAB63634.1|(AC001645) jasmonate inducibleprotein isolog [Arabidopsis thaliana] AC004684.165 17907_s_atgb|AAC23645.1|(AC004684) unknown protein [Arabidopsis thaliana]AC004683.79 16461_i_at gb|AAC28766.1|(AC004683) peroxidase [Arabidopsisthaliana] A71588.1 14015_s_at emb|CAB42586.1|(A71588) unnamed proteinproduct [Arabidopsis thaliana] Upregulated in root with cold stressAL035538.245 16514_at emb|CAB37548.1|(AL035538) putative protein[Arabidopsis thaliana] AF098630.3 19118_s_at emb|CAB41725.1|(AL049730)putative cell wall-plasma membrane disconnecting CLCT protein (AIR1A)[Arabidopsis thaliana] AC007584.48 20194_at gb|AAD32907.1|AC007584_5(AC007584) unknown protein [Arabidopsis thaliana] X74514.2 20238_atemb|CAA52620.1|(X74515) beta-fructofuranosidase [Arabidopsis thaliana]AL049730.104 18983_s_at emb|CAB41721.1|(AL049730) pEARLI 1-like protein[Arabidopsis thaliana] X67421.3 16489_at emb|CAA47807.1|(X67421) extA[Arabidopsis thaliana] AC007060.34 19840_s_at gb|AAD25759.1|AC007060_17(AC007060) Strong similarity to F19I3.2 gi|3033375 putative berberinebridge enzyme from Arabidopsis thaliana BAC gb|AC004238. EST gb|R90518comes from this gene. Acute (3 hr) manitol stress response downregulatedroot genes AC006577.16 12778_r_at “gb|AAD25772.1|AC006577_8 (AC006577)Belongs to the PF|00657 Lipase/Acylhydrolase with GDSL-motif family.ESTs gb|T44453, gb|T04815, gb|T45993, gb|R30138, gb|AI099570 andgb|T22281 come from this gene. [Arabidopsis thaliana]” X98808.1 15985_atemb|CAA67340.1|(X98808) peroxidase ATP3a [Arabidopsis thaliana] ATU5732015137_s_at gb|AAB47973.1|(U57320) blue copper-binding protein II[Arabidopsis thaliana] U81294.2 20421_at emb|CAB10242.1|(Z97336) germinprecursor oxalate oxidase [Arabidopsis thaliana] Z97338.321 16045_s_atemb|CAB10318.1|(Z97338) HSR201 like protein [Arabidopsis thaliana]ATU62330 15623_f_at dbj|BAA21503.1|(D86591) inorganic phosphatetransporter [Arabidopsis thaliana] AC006577.16 12779_f_at“gb|AAD25772.1|AC006577_8 (AC006577) Belongs to the PF|00657Lipase/Acylhydrolase with GDSL-motif family. ESTs gb|T44453, gb|T04815,gb|T45993, gb|R30138, gb|AI099570 and gb|T22281 come from this gene.[Arabidopsis thaliana]” X98855.2 16028_at emb|CAA67361.1|(X98855)peroxidase ATP8a [Arabidopsis thaliana] AF128395.12 20395_at“gb|AAD17355.1|(AF128395) contains similarity to pathogenesis-relatedprotein 1 precursors and SCP-like extracellular proteins (Pfam: PF00188,Score = 79.8, E = 4.1e−21, N = 1) [Arabidopsis thaliana]” Z97340.34517485_s_at “emb|CAB10405.1|(Z97340) beta-1,3-glucanase class I precursor[Arabidopsis thaliana]” ATU10034 15120_s_at gb|AAA93132.1|(U10034)glutamate decarboxylase [Arabidopsis thaliana] AC004521.114 19195_atgb|AAC16105.1|(AC004521) unknown protein [Arabidopsis thaliana] X98319.216971_s_at emb|CAA66963.1|(X98319) peroxidase [Arabidopsis thaliana]X98322.2 17942_s_at emb|CAA66966.1|(X98322) peroxidase [Arabidopsisthaliana] U81294.2 20422_g_at emb|CAB10242.1|(Z97336) germin precursoroxalate oxidase [Arabidopsis thaliana] AL049730.104 18983_s_atemb|CAB41721.1|(AL049730) pEARLI 1-like protein [Arabidopsis thaliana]ATPIN2 12932_s_at gb|AAC84042.1|(AF087459) polar-auxin-transport effluxcomponent AGRAVITROPIC 1 [Arabidopsis thaliana] X67421.3 16489_atemb|CAA47807.1|(X67421) extA [Arabidopsis thaliana] AC004683.7916461_i_at gb|AAC28766.1|(AC004683) peroxidase [Arabidopsis thaliana]AC004005.104 19390_at gb|AAC23409.1|(AC004005) unknown protein[Arabidopsis thaliana] AC004521.119 20608_s_at gb|AAC16106.1|(AC004521)hypothetical protein [Arabidopsis thaliana] Manitol stress responseupregulated in root genes only (acute response) AL080253.32 19415_atemb|CAB45805.1|(AL080253) putative protein [Arabidopsis thaliana]A71596.1 14016_s_at emb|CAB42592.1|(A71596) unnamed protein product[Arabidopsis thaliana] AC001645.19 15965_at gb|AAB63631.1|(AC001645)jasmonate inducible protein isolog [Arabidopsis thaliana] A71588.114015_s_at emb|CAB42586.1|(A71588) unnamed protein product [Arabidopsisthaliana] AC002333.199 13552_at gb|AAB64045.1|(AC002333) endochitinaseisolog [Arabidopsis thaliana] X74514.2 20238_at emb|CAA52620.1|(X74515)beta-fructofuranosidase [Arabidopsis thaliana] AC001645.47 15996_atgb|AAB63634.1|(AC001645) jasmonate inducible protein isolog [Arabidopsisthaliana] ATAJ2596 16085_s_at emb|CAB16787.1|(Z99707) patatin-likeprotein [Arabidopsis thaliana] AC007135.23 20176_atgb|AAD26967.1|AC007135_3 (AC007135) unknown protein [Arabidopsisthaliana] X98320.2 18312_s_at emb|CAA67310.1|(X98774) peroxidase ATP6a[Arabidopsis thaliana] Z99707.288 18326_s_at emb|CAB16788.1|(Z99707)patatin-like protein [Arabidopsis thaliana] BCHI 13211_s_atdbj|BAA82825.1|(AB023463) basic endochitinase [Arabidopsis thaliana]AC005560.147 12758_at gb|AAC67329.1|(AC005560) putative major latexprotein [Arabidopsis thaliana] AL049500.57 16914_s_atemb|CAB39936.1|(AL049500) osmotin precursor [Arabidopsis thaliana]AB023448.2 12332_s_at dbj|BAA82810.1|(AB023448) basic endochitinase[Arabidopsis thaliana] AL035538.245 16514_at emb|CAB37548.1|(AL035538)putative protein [Arabidopsis thaliana] AC007584.48 20194_atgb|AAD32907.1|AC007584_5 (AC007584) unknown protein [Arabidopsisthaliana] Salt stress acute respone down regulated root only AC006577.1612778_r_at “gb|AAD25772.1|AC006577_8 (AC006577) Belongs to the PF|00657Lipase/Acylhydrolase with GDSL-motif family. ESTs gb|T44453, gb|T04815,gb|T45993, gb|R30138, gb|AI099570 and gb|T22281 come from this gene.[Arabidopsis thaliana]” ATU57320 15137_s_at gb|AAB47973.1|(U57320) bluecopper-binding protein II [Arabidopsis thaliana] X98808.1 15985_atemb|CAA67340.1|(X98808) peroxidase ATP3a [Arabidopsis thaliana] U81294.220421_at emb|CAB10242.1|(Z97336) germin precursor oxalate oxidase[Arabidopsis thaliana] Z97338.321 16045_s_at emb|CAB10318.1|(Z97338)HSR201 like protein [Arabidopsis thaliana] X98855.2 16028_atemb|CAA67361.1|(X98855) peroxidase ATP8a [Arabidopsis thaliana]AC006577.16 12779_f_at “gb|AAD25772.1|AC006577_8 (AC006577) Belongs tothe PF|00657 Lipase/Acylhydrolase with GDSL-motif family. ESTsgb|T44453, gb|T04815, gb|T45993, gb|R30138, gb|AI099570 and gb|T22281come from this gene. [Arabidopsis thaliana]” X78586.2 16048_atemb|CAA55323.1|(X78586) Dr4 [Arabidopsis thaliana] ATU62330 15623_f_atdbj|BAA21503.1|(D86591) inorganic phosphate transporter [Arabidopsisthaliana] AC005560.136 16016_at gb|AAC67328.1|(AC005560) putative majorlatex protein [Arabidopsis thaliana] AF098630.3 19118_s_atemb|CAB41725.1|(AL049730) putative cell wall-plasma membranedisconnecting CLCT protein (AIR1A) [Arabidopsis thaliana] AF128395.1220395_at “gb|AAD17355.1|(AF128395) contains similarity topathogenesis-related protein 1 precursors and SCP-like extracellularproteins (Pfam: PF00188, Score = 79.8, E = 4.1e−21, N = 1) [Arabidopsisthaliana]” Z97340.345 17485_s_at “emb|CAB10405.1|(Z97340)beta-1,3-glucanase class I precursor [Arabidopsis thaliana]”AL035538.245 16514_at emb|CAB37548.1|(AL035538) putative protein[Arabidopsis thaliana] X98322.2 17942_s_at emb|CAA66966.1|(X98322)peroxidase [Arabidopsis thaliana] ATU10034 15120_s_atgb|AAA93132.1|(U10034) glutamate decarboxylase [Arabidopsis thaliana]AL049730.104 18983_s_at emb|CAB41721.1|(AL049730) pEARLI 1-like protein[Arabidopsis thaliana] AJ133036.5 15969_s_at emb|CAA67313.1|(X98777)peroxidase ATP16a [Arabidopsis thaliana] U72155.2 15954_atgb|AAB64244.1|(U72155) beta-glucosidase [Arabidopsis thaliana] X98319.216971_s_at emb|CAA66963.1|(X98319) peroxidase [Arabidopsis thaliana]U81294.2 20422_g_at emb|CAB10242.1|(Z97336) germin precursor oxalateoxidase [Arabidopsis thaliana] X67421.3 16489_at emb|CAA47807.1|(X67421)extA [Arabidopsis thaliana] ATPIN2 12932_s_at gb|AAC84042.1|(AF087459)polar-auxin-transport efflux component AGRAVITROPIC 1 [Arabidopsisthaliana] AC005310.6 17697_at gb|AAC33493.1|(AC005310) unknown protein[Arabidopsis thaliana] AC007135.23 20176_at gb|AAD26967.1|AC007135_3(AC007135) unknown protein [Arabidopsis thaliana] Salt stress acuterespone up regulated root only AC005967.50 17864_atgb|AAD03387.1|(AC005967) unknown protein [Arabidopsis thaliana]AC007060.34 19840_s_at gb|AAD25759.1|AC007060_17 (AC007060) Strongsimilarity to F19I3.2 gi|3033375 putative berberine bridge enzyme fromArabidopsis thaliana BAC gb|AC004238. EST gb|R90518 comes from thisgene. BCHI 13211_s_at dbj|BAA82825.1|(AB023463) basic endochitinase[Arabidopsis thaliana] AC001645.19 15965_at gb|AAB63631.1|(AC001645)jasmonate inducible protein isolog [Arabidopsis thaliana] AB023448.212332_s_at dbj|BAA82810.1|(AB023448) basic endochitinase [Arabidopsisthaliana] AC001645.47 15996_at gb|AAB63634.1|(AC001645) jasmonateinducible protein isolog [Arabidopsis thaliana] AL049500.57 16914_s_atemb|CAB39936.1|(AL049500) osmotin precursor [Arabidopsis thaliana]AC007584.48 20194_at gb|AAD32907.1|AC007584_5 (AC007584) unknown protein[Arabidopsis thaliana] Genes expressed in root that have no acuteresponse to stress X98321.2 19595_s_at emb|CAA66965.1|(X98321)peroxidase [Arabidopsis thaliana] AC006216.26 18571_atgb|AAD12681.1|(AC006216) Similar to gi|3413714 T19L18.21 putativemyrosinase-binding protein from Arabidopsis thaliana BAC gb|AC004747.ESTs gb|65870 and gb|T20812 come from this gene. AC006216.22 14050_at“gb|AAD12679.1|(AC006216) Similar to gi|3413714 T19L18.21 putativemyrosinase-binding protein from Arabidopsis thaliana BAC gb|AC004747.ESTs gb|T44298, gb|T42447, gb|R64761 and gb|I100206 come from thisgene.” AL080253.32 19415_at emb|CAB45805.1|(AL080253) putative protein[Arabidopsis thaliana] X74514.2 20239_g_at emb|CAA52620.1|(X74515)beta-fructofuranosidase [Arabidopsis thaliana] AC002333.210 13153_r_atgb|AAB64320.1|(AC002335) endochitinase isolog [Arabidopsis thaliana]CAFFEROYLCOAMETHYLTRANS 13215_s_at gb|AAA62426.1|(L40031)S-adenosyl-L-methionine: trans- caffeoyl-Coenzyme A3-O-methyltransferase [Arabidopsis thaliana] ATHORF 16649_s_atgb|AAA62426.1|(L40031) S-adenosyl-L-methionine: trans- caffeoyl-CoenzymeA 3-O-methyltransferase [Arabidopsis thaliana] AC003673.201 16481_s_atgb|AAC09031.1|(AC003673)peroxidase ATP22a [Arabidopsis thaliana]AL049638.193 20029_at emb|CAB40949.1|(AL049638) putative DNA-bindingprotein [Arabidopsis thaliana]

2. Dynamics of Gene Expression During Leaf Development

In order to examine the dynamics of gene expression at mRNA level duringdifferent organ development, genes with transcripts detected in variousdevelopmental stages were analyzed. A total of 5,247 genes expressedduring leaf development were subject to cluster analysis. Variousclustering methods, including self-organizing map (SOM, Tamayo et al.,1999), hierarchical cluster (Eisen et al., 1998) and K-mean, generatedsimilar clusters. Sixteen groups of genes formed according to theirexpression patterns when SOM was used. Four groups of genes wereexamined in detail.

Cluster 15 shows a group of genes down regulated during leafdevelopment. Genes in this group generally have a very hightranscription level. However, they reduce their expression level byleast 2-fold toward senescence. Among 34 genes in the cluster, 28 ofthem were photosynthesis related. Interestingly, some of the genesrelated to photosynthesis are also found in cluster 6, which shows amore gradual reduction in expression. These genes, such asferredoxin-NADP+reductase and NADPH protochlorophyllide oxidoreductaseB, have relatively low level of transcripts, and their reduction is notas dramatic as others.

Cluster 8 was also analyzed. The expression of this group of genes showsa dramatic increase towards senescence. Detailed examination of thiscluster revealed 8 genes involved in senescence. Other senescence genesalso increased their transcription level during late development,however, those changes were not as dramatic as the eight genesidentified in cluster 8. These genes were found in cluster 2.

3. Function Characterization of Global Gene Expression Pattern

Cluster analysis also identifies co-regulated genes, and organizes thesamples or array experiments according to their overall expressionpatterns. In order to validate the expression data, cluster analysis wasperformed on 6,626 genes with an expression level above background(average difference greater or equal 25) in any of the samples. All datawere normalized to their median, organized into a SOM, and into ahierarchical cluster using Cluster program (Eisen et al. 1998).

According to the similarity of the global expression patterns of eachsample, samples form three major clusters: a cluster of leaf samples, acluster of supporting axis, including root, inflorescence stem andseedling samples, and a cluster of the reproductive organ samples,including samples of flowers, siliques, and inflorescences (includingflowers and siliques). Similarly, genes also organized into severalmajor classes according to their expression levels: organ-differentiallyexpressed genes were easily highlighted.

It is worth noting that sample/experimental variations also contributedto the clusters. For example, the leaf gene expression data wereproduced from 2 independent experiments. One set of the leaf materialswas collected in the morning at approximately 10 o'clock, and the otherset was collected in the afternoon around 3 o'clock in the afternoon.The circadian regulated gene expression contributed greatly to form twosample clusters. These circadian regulated genes matched the genesdescribed in Hammer et al. (2000).

4. Regulatory Sequences

To elucidate the regulatory elements of co-regulated genes, AlignACE wasemployed (Hughes et al., 2000). A total of 49 promoters were found toshare a few potential and known cis-acting elements. Among thesecis-acting elements identified from the ribosomal promoters, thetelo-box motif (AAACCCTA) was observed in 41 of these ribosomalpromoters. Telo-boxes have been found in many Arabidopsis ribosomalgenes and in eEF1A (Tremousaygue et al., 1999). This telo-box binds aprotein related to Pura conserved nuclear protein that has beenimplicated in the control of gene transcription and DNA replication(Safak et al., 1999). Another motif identified in the ribosomal promoterregions was the D of binding site (AAAG). The D of binding site has beenshown in the promoters of a diverse set of plant genes, suggestingvarious roles of D of proteins in plants (Yanagisawa and Schmidt, 1999),including carbon metabolism (Yanagisawa, 2000). Additional motifsobserved include a pollen specific motif (AGAAA) and a RAV1 bindingmotif (Kagaya et al., 1999).

The promoter regions from leaf-specific genes were also analyzed byAlignAce software to discover putative cis elements. Those that werefound include a GATA box and a light regulatory element “ACGTGGCA”.These elements are known to be necessary for light induced genes. Aputative element that did not contain a known binding site was“TGGTTCGGACC” (SEQ ID NO:28).

A global gene expression pattern composed of the transcription profilesof 8,100 genes in 20 samples collected from different organs duringArabidopsis development was identified. By 166,000 gene expressionmeasurements, the mRNA populations in different organs duringArabidopsis development were characterized. In particular,constitutively expressed genes and organ-differentially expressed geneswere identified.

The accuracy of the microarray data was validated by two measures.First, the microarray results were repeatable. By comparing 15 pair ofindependently prepared labeled samples, less than 0.2% of the falsepositive rate was observed. The false positives occurred randomly amongthe genes with a low expression level. Second, expression levelsmeasured by the oligonucleotide array correlated well with data fromprevious gene expression analysis and measurement from othertechnologies, such as RT-PCR.

Identification of constitutively and organ-differentially expressedgenes is important to isolate constitutive or organ/tissue specificpromoters. Here, it is demonstrated that the microarray technology canbe used for large scale screening of these promoters, especially at thegenome level. Moreover, genes that are co-regulated can be analyzed toidentify the regulatory elements. In this study, constitutive andorgan-specific genes were identified through the screening of 8,100genes, but also regulatory elements, such as telo-box, D of bindingsite, as well as other motifs, which are important for the constitutiveexpression of the ribosomal proteins. By a similar approach, organ- ortissue-specific gene promoter elements, and various treatment-inducedgene promoter elements, have been identified. Such results not onlyfacilitate the dissection of the regulatory pathway, but also provide anopportunity in genetic engineering of metabolic pathways. Methods suchas chimeraplasty (Zhu et al. 1999, 2000) can be used to precisely modifythese regions and thus regulate a group of genes of interest.

Identification of co-regulated genes is the first step towardsunderstanding of the regulation of a gene expression network, andassigning function to new genes. Among the 8,100 genes analyzed,approximately 3,100 genes do not have significant homology to knowngenes. Functional characterization of these genes becomes the challengefor the Arabidopsis genomics. A straightforward approach can be used toassign gene function: mutant lines or treated biological samples andtheir controls can be transcriptionally profiled. By comparingalterations in the expression of the novel genes, potential function canbe assigned. The functions can be further confirmed by reverse genetics.Alternatively, genes with unknown function in the identifiedco-regulated gene clusters can be computationally analyzed by supportvector machines (SVMs; Brown et al. 2000).

Similar experiments were conducted for root at least 3 hours afterexposure to stress, e.g., salt, mannitol or cold (Tables 7-8).Twenty-five root-specific promoters were downregulated and 8 wereupregulated in response to salt stress, 21 were down-regulated and 17were upregulated in response to mannitol, and 22 were downregulated and7 were upregulated in response to cold. Ten promoters did not respond toany of the stresses.

Example 3 Further Analysis of Constitutively Expressed Genes

A standard curve of 50, 10, 2, 0.4, and 0.08 ng total RNA was generatedfor each primer/probe set tested. In this case, the 50 ng sample yieldeda C_(t) value of 24.5 and the ng sample yields a C_(t) value of 26.7.The C_(t) value is defined as the threshold cycle whereby amplificationoccurs at an exponential rate. A low C_(t) value correlates with highgene expression. The threshold is determined empirically from thestandard curve. By raising or lowering the threshold, the data set ismaximized to represent optimal exponential amplification. A correlationcoefficient (R² of the best-fit line from the standard curve) greaterthan 99% and a slope of −3.3 (most efficient amplification) is ideal.For accurate repeatable results, the previous criteria must be met andthe unknowns must fall within the range of the curve. The expressionlevels of the unknown can be interpolated from the unknown C_(t) valuesusing the standard curve.

TaqMan chemistry employs three gene-specific oligonucleotides for thedetection of nucleic acids. Two of the oligonucleotides are primers usedfor the amplification of the molecule and the third oligonucleotide is aprobe that is labeled with a 5′ fluorescent reporter dye (6-FAM) and a3′ quencher dye (TAMRA). During PCR amplification, elongation proceedsonce the DNA polymerase binds to the primer. As it polymerizes in the 5′to 3′ direction, the polymerase encounters the quenched probe. The 5′ to3′ exonuclease activity of the polymerase allows it to degrade the probein its path, thereby releasing the 5′ reporter dye. The thermocycler isequipped with a detection system to measure the fluorescence from thereleased reporter dye. Since fluorescence increases with amplificationof the molecule, fluorescence can be directly related to the amount ofmolecules in the starting sample. The primers that were employed for oneset were: TRX3T 5′ 6-FAM agacttcactgcaacatggtgcccac TAMRA 3′ (SEQ IDNO:29); TRX3F 5′ gtgtggaaatgacacagattgtga3′ (SEQ ID NO:30), and TRX3R 5′agacgggtgcaatgaaacg3′ (SEQ ID NO:31); and for the other set were: APX3 T5′ 6-FAM cgcgaacaagaactgtgctcctatcatg TAMRA 3′ (SEQ ID NO:32), APX3 F 5′gccgtgagctccgttctct3′ (SEQ ID NO:33); and APX3 R 5′ tcgtgccatgccaatcg3′(SEQ ID NO:34). TaqMan chemistries were used with the ABI Prism 7700Sequence system for relative quantitation of nucleic acid.

To find a gene whose expression is constitutive, the gene expressiondata obtained from the Arabidopsis GENECHIP® was analyzed. Three sets ofdata were analyzed (Table 9). Part A represents expression data for 2genes from wild-type plants infected or not infected with Pseudomonassyringae pv. maculicola strain ES4326 at 30 hours post-inoculation. PartB represents expression data from wild-type Arabidopsis plants infectedor not infected with 5 different viruses at 1 and 4 days afterinoculation, while part C represents expression data for 2 genes in 9different tissue types.

TABLE 9 A: PLANTS TRX3 APX3 Columbia infected 2481 484 Columbia mock2362 495 B: DAYS GENE Mock TVCV ORMV TRV CMV TuMV 1 TRX3 2020 1991 17382006 1833 1867 1 APX3 711 557 717 755 658 426 4 TRX3 1753 1978 1377 22491918 1928 4 APX3 759 674 428 551 741 434 C: TRX3 APX3 4 day seed 1282488 2 week root 1467 435 Adult root 1857 320 2 week leaf 1233 771 Adultleaf 1483 857 Senescing leaf 1312 805 Flowers 694 513 Inflorescence 691461 Immature siliques 614 508

After analyzing the data, 2 candidate genes were identified, thioredoxin(TRX3; GENBANK® Accession No. U35640) and ascorbate peroxidase (APX3;GENBANK® Accession No. U69138), whose expression did not vary more than2-fold between the treatments in all experiments (except in flowers,inflorescence and siliques for TRX3). These genes also met the criteriaof not having significant sequence similarity to other Arabidopsisgenes.

Probe and primer sets were prepared for ubiquitin 5 (UBQ5), PR1 (apathogenesis related gene whose expression is induced upon infection),TRX3 and APX3. TaqMan was used to quantify relative expression levels ofthese genes in Arabidopsis mutants and in uninfected and P. syringaeinfected plants. Table 10 shows that the PR1 expression increasedrapidly upon infection. TRX3 and APX3 expression levels did not changeas much as UBQ5, a commonly used gene for normalization.

TABLE 10 Gene expression in Arabidopsis infected with P. syringae at 34hours post inoculation. Measured by TaqMan. PLANTS PR1 UBQ5 TRX3 APX3Columbia 10 15 1.2 1.4 infected Columbia .0033 2.7 .62 1.4 Mock Pad4mutant 4.6 2.0 1.2 1.4 infected Pad4 mutant .00027 .79 1.1 2 MockAdditionally, Arabidopsis plants were cold treated for 48 hours and thegene expression of these plants versus plants left at room temperaturemeasured. There was no significant gene expression difference for PR1,TRX3, or APX3 (Table 11).

TABLE 11 Room temperature plants Cold-treated plants PR1 2.6 3.2 TRX32.0 2.4 APX3 2.1 2.8

In summary, gene-chip data was employed to find genes whose expressionis constitutive in several Arabidopsis mutants, in infected plants, andthroughout different tissues. TRX3 and APX3 expression levels variedless than UBQ5 in a comparison between infected and uninfected plants.TRX3 and APX3 gene expression was not significantly affected bycold-stress. Thus, TRX3 and APX3 are candidates for normalization whendetermining unknown gene expression levels in plants such as Arabidopsisor using quantitative PCR or other gene expression measurement assays.Likewise, the plant kingdom orthologs of these genes in dicots andmonocots can be used for the same normalization standards for plantsunrelated to Arabidopsis.

Moreover, unlike actin and ubiquitin (actin mediates cellular divisionand cycling and the ubiquitin pathway is activated upon stress, all ofwhich may result in changes in gene expression), which belong to genefamilies to which probes can cross-hybridize, TRX3 and APX3 genes do nothave significant similarity to genes in the Arabidopsis genome database,and the respective primer/probe sets described herein did notsignificantly cross-hybridize with other genes in the Arabidopsis genomedatabase. Additionally, the promoters for these genes may be useful forconstitutive gene expression.

Example 4 Construction of Binary Promoter::Reporter Plasmids

To construct a binary promoter::reporter plasmid for Arabidopsistransformation a vector containing a promoter of interest (i.e., the DNAsequence 5′ of the initiation codon for the gene of interest) was used,which resulted from recombination in a BP reaction between a PCR productusing the promoter of interest as a template and pDONRneo. Theregulatory/promoter sequence was fused to the GUS reporter gene(Jefferson et al, 1987) by recombination using GATEWAY™ Technologyaccording to manufacturers protocol as described in the InstructionManual (GATEWAY™ Cloning Technology, GIBCO BRL, Rockville, Md.).Briefly, the promoter fragment in the vector is recombined via the LRreaction with a binary Agrobacterium destination vector containing theGUS coding region with an intron that has an attR site 5′ to the GUSreporter (pNOV2374). The orientation of the inserted fragment wasmaintained by the att sequences and the final construct was verified bysequencing. The construct was then transformed into Agrobacteriumtumefaciens strains by electroporation.

pNOV2374 is a binary vector with a VS1 origin of replication, a copy ofthe Agrobacterium virG gene in the backbone and a Basta resistanceselectable marker cassette between the left and right border sequencesof the T-DNA.

The Basta selectable marker cassette comprises the Agrobacteriumtumefaciens manopine synthase promoter (AtMas et al., 1983) operablylinked to the gene encoding Basta resistance (denoted here as “BAR”,phosphinothricin acetyl transferase, White et al, 1990) and the 35Sterminator. The AtMas promoter, BAR coding sequence and 35S terminatorare located at nt 4211 to 4679, nt 4680 to 5228, and nt 5263 to 5488,respectively, of pNOV2374. The vector contains GATEWAY™ recombinationcomponents which were introduced into the binary vector backbone byligating a blunt-ended cassette containing attR sites, ccdB andchloramphenicol resistance marker using the GATEWAY™ Vector ConversionSystem (LifeTechnologies). The GATEWAY™ cassette is located between nt126 and 1818 of pNOV2374. The promoter cassettes are inserted through anLR recombination reaction whereby the DNA sequence of pNOV2374 betweennt 126 and nt 1818 are removed and replaced with the promoter ofinterest flanked by att sequences. The recombination results in thepromoter sequence fused to the GUS reporter gene with intron (GIG)sequence. The GIG gene contains the ST-LS1 intron from Solanum tuberosumat nt 385 to nt 576 of GUS (obtained from Dr. Stanton Gelvin, anddescribed in Narasimhulu et al, 1996).

For comparison of promoter activity an additional construct was producedwith the known Arabidopsis ubiquitin 3 (Ubq3(At), (Callis et al., 1990)promoter plus intron operatively linked to the GIG gene and the nospromoter. Thus, the orientation of the selectable marker andpromoter-reporter cassette in the binary vector construct was RBUbq3(At) promoter with intron fragment+GIG gene+nos—AtMas+BAR+35S ter—LB

Example 5 In Vitro Promoter Assays and Arabidopsis Transformation PlantPreparation and Growth

Arabidopsis seeds are sown on moistened Fafard Germinating Mix at adensity of 9 seeds per 4″ square pot, placed in a flat, covered with aplastic dome to retain moisture and moved to a growth chamber. Followinggermination the dome is removed and plants are grown for 3-5 weeks undershort days (8 hrs light) to encourage vegetative growth and productionof large plants with many flowers. Flowering is induced by providinglong days (16 hrs. light) for 2-3 weeks, at which time plants are readyfor dip inoculation into Agrobacterium to generate transgenic plants.

Agrobacterium Transformation, Culture Growth and Preparation for PlantInfiltration

The binary promoter::reporter plasmids are introduced into Agrobacteriaby electroporation. The binary plasmid confers spectinomycin resistanceto the bacteria allowing cells containing the plasmid to be selected bygrowth of colonies on plates of LB+spectinomycin (50 mg/L). Presence ofthe correct promoter::GUS plasmid is confirmed by sequence analysis ofthe plasmid DNA isolated from the bacteria.

Two days prior to plant transformation 5 mL cultures of LB+spectinomycin(50 mg/L) are inoculated with the Agrobacterium strain containing thebinary promoter::GUS plasmid and incubated at 30° C. for about 24 hours.Each 5 mL culture is then transferred to 500 mL of LB+spectinomycin (50mg/L) and incubated for about 24 hours at 30° C. Each 500 mL culture istransferred to a centrifuge bottle and centrifuged at 5000 rpm for 10minutes in a Sorvall Centrifuge. The supernatant is removed and thepelleted Agrobacterium cells are retained. The Agrobacterium cells areresuspended in 500 mL of modified Infiltration Media (IM+MOD: 50 g/Lsucrose, 10 mM MgCl, 10 uM benzylaminopurine) to which 50 ul of SilwetL-77 (Dupont) has been added.

Plant Transformation by Dip Infiltration

Resuspended cells are poured into 1 L tri-pour beakers. Flowering plantsare inverted into the culture, making sure all inflorescences arecovered with the bacteria. The beakers are gently agitated for 30seconds, keeping all inflorescence tissue submerged. Plants are returnedto growth chamber following dip inoculation of the Agrobacterium. Asecond dip may be performed 5 days later to increase transformationfrequency. Seeds are harvested ˜4 to 6 weeks after transformation.

Selection of Transgenic Arabidopsis

Seeds from transformed Arabidopsis plants are sown on moistened FafardGerminating Mix in a flat, covered with a dome to retain moisture andplaced in a growth chamber. Following germination seedlings are sprayedwith the herbicide BASTA. Transgenic plants are BASTA resistant due tothe presence of the BAR gene in the binary promoter::GUS plasmid.

Promoter Assays

Promoter activity is evaluated qualitatively and quantitatively usinghistochemical and florescence assays for expression of theβ-glucuronidase (GUS) enzyme.

Histochemical β-Glucuronidase (GUS) Assay

For qualitative evaluation of promoter activity, various Arabidopsistissues and organs are used in GUS histochemical assays. Either wholeorgans or pieces of tissue are dipped into GUS staining solution. GUSstaining solution contains 1 mM 5-bromo-4-chloro-3-indolyl glucuronide(X-Gluc, Duchefa, 20 mM stock in DMSO), 100 mM Na-phosphate buffer pH7.0, 10 mM EDTA pH 8.0, and 0.1% Triton X100. Tissue samples areincubated at 37° C. for 1-16 hours. If necessary samples can be clearedwith several washes of 70% EtOH to remove chlorophyll. Followingstaining tissues are viewed under a light microscope to evaluate theblue staining showing the GUS expression pattern.

β-Glucuronidase (GUS) Florescence Assay

For quantitative analysis of promoter activity in various Arabidopsistissues and organs, GUS expression is measured fluorometrically. Tissuesamples are harvested and ground in ice cold GUS extraction buffer (50mM Na₂HPO₄ pH 7.0, 5 mM DTT, 1 mM Na₂EDTA, 0.1% Triton X100, 0.1%sarcosyl). Ground samples are spun in a microfuge at 10,000 rpm for 15minutes at 4° C. Following centrifugation the supernatant is removed forGUS assay and for protein concentration determination.

To measure GUS activity the plant extract is assayed in GUS assay buffer(50 mM Na₂HPO₄ pH 7.0, 5 mM DTT, 1 mM Na₂EDTA, 0.1% TritonX100, 0.1%sarcosyl, 1 mM 4-Methylumbelliferyl-beta-D-glucuronic acid dihydrate(MUG)), prewarmed to 37° C. Reactions are incubated and 100 uL aliquotsare removed at 10 minute intervals for 30 minutes to stop the reactionby adding to tubes containing 900 uL of 2% Na₂CO₃. The stopped reactionsare then read on a Tecan Spectrofluorometer at 365 nm excitation and 455emission wavelengths. Protein concentrations are determined using theBCA assay following manufacturers protocol. GUS activity is expressed asrelative fluorometric units (RFU)/mg protein.

Example 6 Determination of the Minimal Promoter Fragment

The full-length promoter sequence as given in SEQ ID Nos: 1-26, or thepromoter orthologs thereof is fused to the β-glucuronidase (GUS) gene atthe native ATG to obtain a chimeric gene cloned into plasmid DNA. Theplasmid DNA is then digested with restriction enzymes to release afragment comprising the full-length promoter sequence and the GUS gene,which is then used to construct the binary vector. This binary vector istransformed into Agrobacterium tumefaciens, which is in turn used totransform Arabidopsis plants (for further details of the binary vectorconstruction see above example 4)

The above plasmid can also be used to form a series of 5′ end deletionmutants having increasingly shorter promoter fragments fused to the GUSgene at the native ATG. Various restriction enzymes are used to digestthe plasmid DNA to obtain the binary vectors with different lengths ofpromoter fragments. In particular, a binary vector 1 is constructed witha 1,900-bp long promoter fragment; a binary vector 2 is constructed witha 1,300-bp long promoter fragment; a binary vector 3 is constructed witha 1000-bp long promoter fragment; a binary vector 4 is constructed witha 800-bp long promoter fragment; a binary vector 5 is constructed with a700-bp long promoter fragment; a binary vector 6 is constructed with a600-bp long promoter fragment; a binary vector 6 is constructed with a500-bp long promoter fragment; and a binary vector 7 is constructed witha 100-bp long promoter fragment. Like the binary vector comprising thefull-length promoter fragment, these 5′ end deletion mutants are alsotransformed into Agrobacterium tumefaciens and, in turn, Arabidopsisplants (for further details of Arabidopsis transformation and promoterassay procedures see example 5 above).

The presence of the correct hybrid construct in the transgenic lines isconfirmed by PCR amplification.

By using the above protocol it can be determined, which portion of thepromoter sequences given in SEQ ID Nos: 1-26, or the promoter orthologsthereof is required for gene expression.

Minimal promoter fragments having lengths substantially less than thefull-length promoter can therefore be operatively linked to codingsequences to form smaller constructs than can be formed using thefull-length promoter. As noted earlier, shorter DNA fragments are oftenmore amenable to manipulation than longer fragments. The chimeric geneconstructs thus formed can then be transformed into hosts such as cropplants to enable at-will regulation of coding sequences in the hosts.

Example 7 Determination of Promoter Motifs

While a deletion analysis characterizes regions in a promoter that arerequired overall for its regulation, linker-scanning mutagenesis allowsfor the identification of short defined motifs whose mutation alters thepromoter activity. Accordingly, a set of linker-scanning mutantpromoters fused to the coding sequence of the GUS reporter gene areconstructed. Each of them contains a 8-10-bp mutation located betweendefined positions and included in a promoter fragment as given in SEQ IDNos: 1-26, or the promoter orthologs thereof.

Each construct is transformed into Arabidopsis and GUS activity isassayed for 19 to 30 independent transgenic lines. The presence of thecorrect hybrid construct in transgenic lines is confirmed by PCRamplification of all lines containing the mutant constructs and byrandom sampling of lines containing the other constructs. Amplifiedfragments are digested with restriction enzyme (e.g. XbaI) and separatedon high resolution agarose gels to distinguish between the differentmutant constructs. The effect of each mutation on promoter activity iscompared to an equivalent number of transgenic lines containing theunmutated construct. Two repetitions resulting from independent platingof seeds are carried out in every case.

The sequences mutated in the linker-scanning constructs, in particularthose that showed marked differences from the control construct, arethen examined more closely.

Example 8 Wound-Inducible Promoter

The AR10 promoter (SEQ ID NO: 17) is a root-specific promoter inArabidopsis. Interestingly, AR10 was found to be a wound-induciblepromoter when in a heterologous transgene.

An expression cassette containing the AR10 promoter was constructed byPCR amplifying the AR10 promoter from Arabidopsis and adding to it aSanDI site at the 5′ terminus and a BamHI-TAAA-NcoI sequence at the 3′terminus. The PCR product was TOPO cloned and its sequence verified. TheAR10 promoter was then digested with SanDI and NcoI and cloned into aplasmid containing a β-glucuronidase (GUS) gene and a Nos terminator atthe same sites, thereby operably linking the AR10 promoter to GUS andNos. The plasmid containing the AR10:GUS:Nos expression cassette wastransformed into soybean using Agrobacterium tumefaciens. Nine TOtransformation events were selected for analysis.

Tissues were harvested from the T0 plants at early flowering and stainedfor GUS expression in roots, stems, petioles, petiole stems, leaves,flowers, meristems, and wounded areas. Plant tissues were dissected,placed in wells of a 12-well plate, and covered with GUS mix (per 100ml: 10 ml of 1M NaPO₄, pH 7; 2 ml of 500 mM EDTA; 10 ml of 5 mMpotassium ferrocyanide; 10 ml of 5 mM potassium ferricyanide; and 1 ml5-Bromo-4-chloro-3-indoxyl beta-D-glucuronide cyclohexylammonium salt(50 mg/ml in DMSO)). Tissues were vacuum infiltrated for 20 minutes at−20 psi and incubated overnight at 37° C.

Incubated tissues were destained and washed 30 minutes each in 20%, 35%,and 50% ethanol, then fixed for 30 minutes in 5% fromalin, 10% aceticacid, 50% ethanol. Tissues were then washed once for 30 minutes in 10%acetic acid, 50% ethanol and then 30 minutes in 70% ethanol. The stainedtissue samples were stored at 4° C. in 70% ethanol. The degree of GUSstaining of harvested tissues was scored by visual comparison. Therelative degree of GUS staining for different tissues from eachtransgenic soybean plant are shown in Table 12.

The data in Table 12 indicate that the AR10 promoter is capable ofdriving expression of a heterologous protein in wounded tissue.Therefore, the AR10 promoter is useful for driving expression of pestcontrol proteins, such as insecticidal toxins from Bacillusthuringensis, or any other protein readily evident to one skilled in theart. A promoter capable of driving an RNAi or an antisense message withthe purpose of manipulating plant or pest gene expression will preventfeeding site formation or maturation of the pest. For pests that elicita wound response from soybean plants, a specific wound induciblepromoter may be preferred over a constitutive promoter in order toexpress pest proteins at only the sites of interaction between the pestand soybean plant tissue, and therefore keep gene-manipulationtechniques from affecting non-wounded portions of the plant.

Any application where a wound responsive specific promoter is requiredor preferred will find this application useful. The skilled person willrecognize that there are other obvious applications for the AR10wound-inducible promoter, such as in other studies of wound causinginteractions, like those caused by herbivores or environmental factors.

TABLE 12 Degree of GUS staining in tissue types. T0 Plant Tissue TypePlant petiole meri- wound- No. root stem petiole stem leaf flower stemed 1 0 2 0 2 4 1 1 4 2 0 ND 0 ND 0 ND ND 0 3 0 6 ND 2 2 0 1 5 4 0 ND 1 21 ND ND 6 5 0 0 0 0 2 0 0 4 6 0 ND 0 ND 0 0 ND 2 7 0 6 1 1 2 2 3 6 8 0 62 3 1 ND 1 5 9 ND ND ND ND ND ND ND ND 0 = no staining, 1 = very lightstaining, 2 = light staining, 3 = medium light staining, 4 = medium darkstaining, 5 = dark staining, 6 = very dark staining, ND = no data

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All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

TABLE 13 Promoter SEQ ID No. Expression Specificity 1B_syn299 1Constitutive 1G2_syn300 2 Constitutive AC11_syn271 3 ConstitutiveAC12_syn272 4 Constitutive AC13_syn273 5 Constitutive AC20_syn278 6Constitutive AC22_syn280 7 Constitutive AC24_syn282 8 ConstitutiveAC26_syn284 9 Constitutive AC31_syn286 10 Constitutive AC34_syn288 11Constitutive AC38_syn290 12 Constitutive AC40_syn292 13 ConstitutiveAC7_syn267 14 Constitutive AC9_syn269 15 Constitutive AF3_syn312 16Fuitless AR10_syn307 17 Inducible by wounding AR13_syn309 18Root-specific AR1_syn301 19 Root-specific AR2_syn302 20 Root-specificAR5_syn303 21 Root-specific AR6_syn304 22 Root-specific AR8_syn305 23Root-specific ATU56929_syn007 24 Constitutive (AC32) PR1_Syn018 25Inducible by SA, NA, BTJ, pathogens UBQ3_Syn016 26 Constitutive

1. An isolated polynucleotide comprising a wound inducible promoter thatdirects transcription of an operably linked heterologous nucleic acidsequence in a plant cell, wherein the wound inducible promoter comprisesSEQ ID NO:
 17. 2. An expression cassette comprising the polynucleotideof claim 1, wherein the heterologous nucleic acid sequence is an openreading frame.
 3. The expression cassette of claim 2, wherein the openreading frame comprises a coding sequence which, when transcribed at thedirection of the polynucleotide, imparts a phenotype selected from thegroup consisting of insect resistance, disease resistance, herbicideresistance, abiotic stress resistance, a modified enzyme expressionprofile, a modified oil content, and a modified nutrient content.
 4. Atransformed plant, the genome of which comprises the expression cassetteof claim
 3. 5. A cell of the transformed plant of claim
 5. 6. Thetransformed plant of claim 6, wherein the plant is a monocot or a dicotplant.
 7. The transformed monocot plant of claim 7 wherein the plant isa cereal plant.
 8. The cereal plant of claim 8 wherein the plant isselected from the group consisting of maize, wheat, rice, sorghum, andbarley.
 9. The transformed dicot plant of claim 7 wherein the dicot isselected from the group consisting of soybean, cotton, canola, andsugarbeet.
 10. An isolated polynucleotide sequence, wherein thepolynucleotide sequence is SEQ ID NO: 17.